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Met de sterk groeiende aantallen zonnepanelen en windmolens maakt Nederland een inhaalslag met de opwekking van duurzame energie in de vorm van elektriciteit. Helaas bestaat er nog geen goede balans tussen vraag en aanbod en is betaalbare opslag van duurzame stroom nog niet vanzelfsprekend. Een mogelijk alternatief is opslag in de vorm van methaan. Daarover meer leren is de kern van het vierjarige onderzoeksproject Biologische Power-to-Gas (Bio-P2G) van lector Jan-Peter Nap.

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.

The functioning of anaerobic bacteria in river sediments is vital for the stability of freshwater ecosystems. The sensitivity of these organisms for pollutants is therefore important for the establishment of valid sediment quality criteria. Acetate is a key intermediate in the carbon cycle and was chosen as a model substrate for the activity of anaerobic bacteria. The effects of pollutant on the anaerobic mineralization of acetate was studied in sediment microcosms. A small amount (0.7 mug/l) of 14 radioactive C labelled acetate was added to bottles with fresh anaerobic river sediment. The acetate was converted methane and 14 radioactive carbondioxide with a half-life of 0.2 - 0.5 hours. Adding a toxicant decreased the mineralization rate of acetate. The IC10 is defined as the concentration which decreases the mineralization rate of acetate with 10%. Addition of mercury or zinc did not inhibit the acetate mineralization. Benzene, pentachlorophenol, 1,2-dichloroethane and chloroform inhibited the acetate mineralization and showed IC10 concentrations of 480, 19, 0.7 and 0.04 mg/kg sediment respectively. Benzene, 1,2-dichloroethane and chloroform are usually considered to be narcotic toxicants and they have a low octanol water partitioning coefficient. The high toxicity of 1,2-dichloroethane and chloroform shows that these compounds do not exhibit minimum toxicity on bacteria under these conditions but perform a specific mode of action. This indicates that sediment quality criteria solely based on tests with aerobic organisms will not always be sufficient to protect the anaerobic processes in the sediment.

Met een indicator gebaseerd op het concept van Pollution-Induced Community Tolerance (PICT) kunnen veldeffecten van milieustressoren worden vastgesteld. Het rapport geeft een samenvatting van de gebruiksmogelijkheden van een microbiele PICT-indicator, van in situ waargenomen relaties tussen PICT en de structuur en het functioneren van ecosystemen alsmede van de beleidsrelevantie van zulke observaties. PICT is het verschijnsel dat blootstelling aan een giftige stof in verandering van de natuurlijke gemeenschap van organismen resulteert. Dit wordt veroorzaakt door fysiologische of genetische aanpassing van soorten en/of vooafsterven van gevoelige soorten en verschijnen van minder gevoelige soorten. Verlies van gevoelige soorten leidt tot verlies van functies. In een aantal gevallen is ook een vermindering van het aantal soorten in verontreinigde ecosystemen gevonden. Bij traditionele ecologische observaties (bijvoorbeeld soorten en aantallen) is het verband tussen de waargenomen verandering en de aanwezige milieustress vaak niet duidelijk als gevolg van natuurlijke variatie en aanwezigheid van andere stressoren. PICT geeft een causaal verband aan tussen een ecologische observatie en aanwezigheid van een giftige stof. Causaliteit tussen de waargenomen verandering en de potentiele stressfactor is een belangrijk voordeel van een PICT indicator. De PICT indicator kan gebruikt worden om vast te stellen of een specifieke verontreiniging effect heeft op het ecosysteem. De beleidsrelevantie van deze indicator bestaat uit de mogelijkheid hem in te zetten voor validatie in het veld van methoden voor risico-schattingen en als instrument voor inschatting van locatiespecifieke risico's van de aanwezigheid van verontreinigingen.

Ultrafiltratie (UF) is een techniek waarmee micro-organismen met behulp van zeer fijnmazige filters uit water kunnen worden verwijderd. Door ultrafiltratie op te nemen als onderdeel van een afvalwaterzuivering worden de aantallen virussen en bacteriën in afvalwater tenminste een miljoen keer verlaagd. Dit blijkt uit onderzoek van het RIVM dat in opdracht van het waterschap Regge en Dinkel en producent Pentair X-flow is uitgevoerd. Schoner afvalwater na ultrafiltratie: In dit onderzoek zijn de aantallen virussen en bacteriën in ongezuiverd afvalwater vergeleken met het afvalwater van de rioolwaterzuiveringsinstallaties te Glanerbrug en Hengelo dat met een UF-filter is behandeld. In het behandelde afvalwater (UF-permeaat) zijn lage aantallen bacterien aangetroffen en konden ziekteverwekkende virussen niet meer worden aangetoond. Lozing UF-permeaat geen nadelige gezondheidseffecten: Vervolgens is onderzocht of een eventuele lozing van het UF-permeaat op het water van de nabijgelegen Voskampvijver (Enschede) gezondheidseffecten veroorzaakt voor kinderen die daarin zwemmen. Deze effecten zijn geschat op basis van de concentraties virussen en bacteriën in het UF-permeaat en in de Voskampvijver. De risico's blijken afhankelijk te zijn van het type microorganisme. Het risico op een infectie door een blootstelling aan entero- en rotavirussen in de vijver, al dan niet aangevuld met UF-permeaat, is zeer klein. Het risico op een infectie door de Campylobacter-bacterie is echter hoog vanwege de hoge aantallen Campylobacter in de Voskampvijver. Deze bacteriën zijn waarschijnlijk afkomstig van uitwerpselen van vogels en staan dus los van de kwaliteit van het UF-permeaat. Op basis van het risico op infectie met Campylobacter wordt zwemmen afgeraden.

In oppervlaktewater en slib in veeteeltrijk gebied komen hoge percentages bacterien voor die resistent zijn tegen een of meerdere antibiotica. Dit blijkt uit verkennend onderzoek van het RIVM. De herkomst van deze bacterien in het onderzochte gebied is in deze studie niet onderzocht. Wel zijn er aanwijzingen dat ten minste een deel van de bacterien afkomstig is uit mest van nabijgelegen veeteeltbedrijven. Onderzoek naar de mate waarin antibioticumresistente bacterien in oppervlaktewater voorkomen is van belang om te kunnen inschatten in hoeverre mensen via het milieu worden blootgesteld aan deze bacterien. Er zijn meerdere oorzaken waardoor antibioticaresistente bacterien in oppervlaktewater terechtkomen. Bijvoorbeeld doordat mest van dieren die met antibiotica zijn behandeld, afspoelt naar het oppervlaktewater. Een andere oorzaak kan zijn dat gedeeltelijk gezuiverd of ongezuiverd afvalwater in oppervlaktewater wordt geloosd, bijvoorbeeld door ziekenhuizen waar mensen zijn behandeld met antibiotica. Als mensen met verontreinigd oppervlaktewater in aanraking komen, zoals tijdens recreatie, kunnen zij worden blootgesteld aan bacterien die resistent zijn tegen een of meerdere antibiotica. Dit brengt mogelijk volksgezondheidrisico's met zich mee omdat deze antibiotica belangrijk kunnen zijn om infecties te behandelen. De risico's kunnen zich op twee manieren manifesteren. Mensen die aan antibioticaresistente bacterien worden blootgesteld, kunnen zelf het risico lopen ziek te worden van deze - moeilijker te bestrijden - bacterien. Daarnaast is het mogelijk dat mensen zelf niet ziek worden van de resistente bacterien maar ze overdragen aan mensen met verminderde weerstand, zoals ziekenhuispatienten. Deze categorie mensen kan hier vervolgens wel ziek van worden.

In 1990-1992 seven regional Public Health Laboratories sent the results of all their routine determinations of antibiotic susceptibility to the RIVM. Each year data from approximately 125000 isolates were collected on floppy disks. During the investigation no striking changes in the level of resistance occurred. Although regional differences in the level of resistance were observed, these may have been due to differences in the methods applied. Escherichia coli, Staphylococcus aureus, enterococci and coagulase-negative staphylococci were the most frequently isolated bacteria. Resistance of Echerichia coli to amoxycillin, doxycycline and trimethoprim was between 20 and 35%. The new cephalosporins, quinolones and aminoglycosides were still active aginst virtually all Enterobacteriaceae. Resistance of Staphylococcus aureus against beta-lactamase-stable beta-lactam antibiotics was rare (a few percent at most). Resistance was most frequent in enterococci and coagulase-negative staphylococci. The number of isolates for each species or group of species and the percentages of resistance have been compared with the results of other investigations. Our results were similar to those of a similar British survey, whereas in the US resistant bacteria were found more frequently. Also in many other European countries the level of resistance appeared higher.

Consumption of water that is contaminated with pathogens still causes high numbers of death and disease. Understanding the factors that influence the dynamic distribution of waterborne pathogens is important, as this will help understanding improvements and possible solutions. Such understanding is particularly important in a developing country like Bangladesh, where large proportions of the population often have little or no access to clean water. Despite the high relevance for public health, few studies currently exists on the fate and transport of pathogens and the so-called Faecal Indicator Bacteria (FIB, e.g. E. coli, enterococci) in (sub)tropical systems. FIB are susceptible to shifts in water flow and quality. The predicted increases in rainfall and floods due to climate change will exacerbate the faecal contamination scenarios. This could be further compounded by the rapid change in socio-economic conditions (population growth, urbanization, sanitation and agricultural management) in the developing countries. Therefore, to reduce future health risks, understanding the influence of changes in socio-economic conditions and climate on microbial dynamics is important. Very few studies have quantified the relationship between the waterborne pathogens/FIB concentrations and climate and socio-economic changes. In this study a process-based model was developed and a scenario analysis was performed based on the new combined climate and socio-economic changes scenarios, to assess the present and future river hydrodynamics, FIB sources, die-off processes and concentrations. We used FIB, because measuring FIB are cheaper than pathogens. FIB are usually not pathogenic but their presence indicates the likely presence of waterborne pathogens. These pathogens are expected to respond to climate change in a comparable way to FIB. The present study is based on the Betna River basin in southwestern Bangladesh, where faecal contamination is not monitored and very little knowledge exists on the distribution of contaminants. First of all, FIB concentrations of the river water were measured to identify the river’s faecal contamination levels that can be used to validate the water-quality model. In the study area, wastewater is not treated and this untreated wastewater is discharged directly into the river. This is evident from the measured FIB data. In 88% of the E. coli and all enterococci samples, the USEPA bathing water quality standards were violated (Chapter 2). Such violation indicates potential health risks associated with the use of the river water for domestic, bathing and irrigation purposes. The correlation between environmental variables (water temperature, precipitation and salinity) and FIB concentrations was also determined. A positive correlation was found with water temperature and precipitation, and a negative correlation with salinity. The positive correlation with temperature is due to the co-occurrence of high summer temperature with abundant monsoon rainfall. The positive correlation with precipitation can be explained by the increased runoff from agricultural lands and urban areas. This runoff contains many bacteria. In the study area, during the rainy season (July to September) precipitation increases and as a result water salinity decreases. The observed negative correlation with salinity is more likely due to the typical weather patterns during the rainy season when low salinity coincides with increased precipitation and high temperature, than to salinity dependent die-off of bacteria. A regression model was applied that explained almost half of E. coli and enterococci variability in river water. This, however, only considers water temperature and precipitation (Chapter 2). Then, the present and future hydrodynamics of the river were simulated using a two dimensional hydrodynamic model (MIKE 21 FM). Although the main goal of this thesis is to assess the river’s present and future FIB concentrations, the reasons for this hydrodynamic modelling are twofold. Firstly, outputs of the hydrodynamic model are used as input into the water-quality model (Chapter 4). Secondly, hydrodynamics (i.e. water level and discharge) are simulated because increased water level and discharge together with sea level rise stimulate floods in the river basin. These floods are related to outbreaks of waterborne diseases. The modelled results corresponded very well with the measured water levels and discharges. The model was applied to simulate baseline and future water levels and discharge for Representative Concentration Pathway RCP4.5 and RCP8.5 scenarios using bias-corrected downscaled data from two climate models (IPSL-CM5A and MPI-ESM). The model results showed an expected increase in water level up to 16% by the 2040s and 23% by the 2090s (Chapter 3). The monsoon daily maximum discharge was expected to increase up to 13% by the 2040s and 21% by the 2090s. These model results also showed that the duration of the water level above the danger level and extreme discharge periods can increase by half a month by the 2040s and over a month by the 2090s. The coincidence of the water danger level with extreme discharge may cause disastrous floods in the study area. Next, the hydrodynamic model was coupled with a water-quality module (ECOLab). The fate and transport of FIB was simulated, the influence of different processes tested and the contribution from different sources to the total contamination quantified (Chapter 4). The model outputs corresponded very well with the measured FIB data. The present river microbial water quality based on measured and simulated results indicated, once again, noncompliance with bathing water standards. Primary and secondary levels of wastewater treatment were not sufficient to reach the standards most of the time, and discharges from sewer drains and incoming concentrations from the upstream boundary were found to be a major cause of water contamination. Tide, wind and diffuse sources (urban and agricultural runoff) contributed little. The high FIB inputs from the upstream open boundary come from untreated point source discharges from upstream urban areas and accumulation of diffuse contaminants from the large upstream areas. Therefore, this study underlines the need for establishment of wastewater treatment plants both in the studied basin and upstream urban areas. This study provides insight into bacterial fate and transport mechanisms, contribution of different sources to the faecal contamination and applicability of wastewater treatment in a river of a subtropical developing country where this type of study is lacking. Uncertainties are related to the lack of high temporal resolution measured FIB data and the lack of available data for contaminant loads from septic tank leakages, open defecation and sediment resuspension. However, the model well captured the measured FIB variability, suggesting that it can be applied for microbial water quality assessments in other watersheds of the world with similar characteristics. The developed model could be an ideal tool to forecast future impacts of climate and socioeconomic changes on FIB fate, transport and dynamics. Finally, future FIB concentrations were simulated using the coupled hydrodynamic and microbial model (MIKE 21 FM-ECOLab) and scenario analysis (Chapter 5). Scenarios have been developed building on the most recent Shared Socio-economic Pathways (SSPs) and Representative Concentration Pathways (RCPs) scenarios from the Intergovernmental Panel on Climate Change (IPCC). We developed a baseline scenario (October 2014–September 2015) reflecting the current conditions and two future scenarios, S1 (sustainability scenario) and S2 (uncontrolled scenario) mimicking different future developments of socio-economic (population, urbanization, sanitation, wastewater treatment development, land use) and climate-change factors (temperature, precipitation and sea-level rise). In S1 RCP4.5 was combined with socio-economic scenarios SSP1, and for S2 RCP8.5 was combined with SSP3 (S2). Assumptions on sanitation, waste water treatment and agricultural management in line with the storylines were made to quantify future changes in FIB concentrations and consequent health risk. Different future scenarios were found to have substantial impact on FIB concentrations in the river. By the 2090s, FIB concentrations are expected to decrease by 98% or increase by 75% for the sustainability scenario and uncontrolled scenario respectively. An uncontrolled future resulted in a deterioration of microbial water quality due to socio-economic developments, such as higher population growth, land-use change and increased sewage discharges and changes in rainfall patterns. Microbial water quality strongly improved under a sustainable climate and improved sewage treatment. FIB concentrations were much more sensitive to changes in socio-economic factors than to changes in climatic factors. This underlines the importance of socio-economic factors in assessing and improving microbial water quality. The results show the importance of improvements in sanitation and wastewater treatment in the Bangladeshi Betna River basin to ensure that future FIB concentrations in the river comply with the US-EPA bathing water quality standards. Major investments to construct wastewater treatment plants are necessary to compensate for the population growth and increased the volume of wastewater treatment. Although the current level of contamination is already too high, without wastewater treatment the water quality will further deteriorate. The thesis assesses the present and future FIB dynamics in the Betna River through sampling, statistical and process-based modelling, and scenario analysis. The results contribute to increase the knowledge base on the dynamic distributions of the FIB in surface water in a developing country and in a subtropical system, where this type of study is lacking. It also reduces the knowledge gaps regarding future flooding scenarios at the local scale. While some earlier studies focused on only assessing climate-change impacts on microbial water quality, this study for the first time assessed the influence of combined climate and socio-economic scenarios (using scenarios based on the new SSP-RCP scenario matrix) on river FIB concentrations. This combined modelling and scenario approach enables the assessment of faecal contamination sources and dynamics at present and in the future. The developed model and scenario analysis approach provides a basis for the water managers to reduce the widespread faecal contamination and the risks of waterborne disease outbreaks, which are still a leading cause of deaths in developing countries.

Sulfur cycle is one of the main geochemical cycles on Earth. Oxidation and reduction reactions of sulfur are mostly biotic and performed by microorganisms. In anaerobic conditions – marine and some freshwater systems, dissimilatory sulfur- and sulfate-reducing bacteria and archaea are key players in the decomposition of organic carbon releasing sulfide as the product of their metabolism. Sulfide can then be used as terminal reductant by anoxygenic photosynthetic microorganisms or it can be used as electron donor for aerobic or nitrate-reducing bacteria, etc. One particular case of the sulfur cycle is the naturally occurring oxidation of metallic sulfide-ores, which produce sulfur-rich waters with low pH and high heavy metals content. Extremophilic sulfur-reducing microorganisms are of scientific and technological interest. They are abundant in natural conditions in extreme environments, so they are environmentally relevant. Although hydrogen sulfide is corrosive and odorous, its production can be beneficial for industrial activities such as the precipitation and recovery of heavy metals. Therefore, sulfur reducers have also potential for extending the range of operating conditions of metal precipitation. This thesis describes the isolation and characterization of acidotolerant sulfur-reducing bacteria, providing a first understanding on their metabolism of sulfur compounds and insights on the beneficial microbial interactions for biotechnological purposes. In Chapter 2 , the ecology and physiology of sulfur-reducing prokaryotes is investigated. The ability of sulfur reduction is wide-spread phylogenetically over the microbial tree of life, found in more than 70 genera. Elemental sulfur reduction can occur via direct cell attachment to the solid substrate or with polysulfide as an intermediate. At least four different enzymes are described to be involved in sulfur reduction pathways, and these enzymes were also detected in several microorganisms that are potential sulfur reducers, but were not reported as such in literature so far. The ecological distribution of sulfur respiration seems to be more widespread at high temperatures with neutral pH values. However, some sulfur reducers can grow at pH as low as 1 and the strategies adopted by microorganisms to face high proton concentrations in the environment were commented in this chapter. The sulfide produced from sulfur reduction can be used to selectively precipitate metals by varying the pH values from 2 to 7, depending on the target metal. Economic calculations were presented to show that sulfur reduction is more advantageous then sulfate reduction due to the cost savings of the electron donor needed. Therefore, acidophilic sulfur reducers are of particular interest for application in selective precipitation and recovery of heavy metals from metalliferous waste streams and the suitable technologies for that purpose are also discussed. Enrichments for sulfur reducers with various electron donors at low pH and mesophilic conditions were performed from sediments of the acidic Tinto river (Spain). A solid-media with colloidal sulfur was developed to facilitate the isolation of true elemental sulfur reducers at low pH. This strategy resulted in the isolation of a sulfur-reducing bacterium, strain TR1, belonging to the Desulfurella genus. The enrichment and isolation procedure were described in Chapter 3 . The growth and activity of the isolate was tested at different pH values, temperature conditions, utilization of electron donors, and growth in the presence of heavy metals in solution. The isolate showed tolerance to metals, and growth in a broad temperature and pH, revealing its feasibility to precipitate and recover heavy metals from acidic wastewater and mining water, without the need to neutralize the water before treatment. In Chapter 4 , the morphological, biochemical and physiological characterization of the isolate is provided, for which the name Desulfurella amilsii TR1 sp. nov. was proposed. D. amilsii is affiliated to the Deltaproteobacteria class showing 97% of 16S rRNA gene identity to the four species described in the Desulfurella genus. In the presence of elemental sulfur, D. amilsii utilized acetate, formate, lactate, pyruvate, stearate, arginine and H2/CO2 as substrates, completely oxidizing them to H2S and CO2. Besides elemental sulfur, thiosulfate was used as an electron acceptor and the isolate also grew in the absence of external electron donor, by disproportionation of elemental sulfur into sulfide and sulfate. The draft genome sequence of Desulfurella amilsii TR1 and a comparative genomic analysis with the members of Desulfurellaceae family are reported in Chapter 5 . Based on average nucleotide identity and in silico DNA hybridization values, D. multipotens and D. acetivorans were revealed to belong to the same species. Reclassification was therefore suggested. Regarding sulfur metabolism, the analysed genomes encode different sulfur-reducing enzymes per genus. Hippea species encode polysulfide reductase and a sulfide dehydrogenase. The analysed genomes of Desulfurella especies do not possess the polysulfide reductase but possess the sulfide dehydrogenase. D. amilsii is the only member of the family encoding sulfur reductase. Since D. amilsii is able to grow at the lowest pH value, this enzyme was suggested to play a role in sulfur reduction when the microorganism grows in acidic conditions. Genes encoding resistance to acidic conditions were reported for all Desulfurellaceae members, countering physiological tests that showed ability to grow at low pH only for D. amilsii and D. acetivorans. Sulfur respiration by D. amilsii was studied in more detail in Chapter 6 , in which the requirement for cell-sulfur interaction at acidic (pH 3.5) and circumneutral (pH 6.5) conditions was evaluated. D. amilsii was shown to benefit from contact with the insoluble substrate, as activity and number of cells decreased when sulfur was sequestered from the medium in dialysis bags of 6-8 kDa pore size. Besides, the abundance of enzymes possibly involved in sulfur respiration, acid resistance and chemolithotrophic growth were investigated by proteomics. Sulfur reductases were not detected in the dataset, but the limitations of the method might leave membrane-bound proteins underrepresented in the study. Different rhodanese-like proteins were detected in high abundance at low and neutral pH, while sulfide dehydrogenase seems to function as a ferredoxin:NADP oxidoreductase. We suggest that the sulfurtransferases might play a key role in sulfur/polysulfide reduction in D. amilsii. Proteomic data also showed that genes involved in acid resistance are constitutively expressed in this microorganism. Some proteins were exclusively detected at low pH, but with very few overlapping with proteins reported to be involved in acid resistance. Moreover, analysis of the proteome revealed the involvement of the hydrogenase HydABC for oxidation of hydrogen during chemolitotrophic growth, as well as the complete pathway for CO2 fixation via the reductive TCA cycle. More aspects of the sulfur metabolism by D. amilsii were investigated in Chapter 7 . Cultures grown on acetate with sulfur or thiosulfate as electron acceptor and cultures grown by disproportionation of elemental sulfur, all at pH 6.5, had their proteomes compared. Rhodanese-like sulfurtransferases were abundant in all the analyzed conditions, with specific differences in the sequences. In sulfur respiration and disproportionation, sulfurtransferases were the only sulfur enzymes detected and so, they are likely to play a central role in the process. The respiration of thiosulfate is likely to happen via a thiosulfate reductase and a dissimilatory sulfite reductase, highly abundant in this specific condition. Analysis on the heterotrophic cultures revealed the ability of D. amilsii to activate acetate to acetyl-CoA via the acetyl-CoA synthetase enzyme and its oxidation via the TCA cycle being this the first report of acetate activation happening via acetyl-CoA synthetase in sulfur-reducing bacteria. The isolation and characterization of another acidotolerant sulfur respirer, Lucifera butyrica strain ALE, and its growth in co-culture with D. amilsii were described in Chapter 8 . L. butyrica was shown to use a wide range of substrate, such as glucose, lactose, ethanol, glycerol glycogen, peptone, etc. When growing on glycerol, a cheap substrate, by fermentation or by respiration of elemental sulfur, L. butyrica produced acetate, ethanol and 1,3-propanediol as major products. Elemental sulfur reduction by this bacterium, however, was not efficient and led to the production of maximum 2.5 mM of sulfide. When L. butyrica grew in a co-culture with D. amilsii, the acetate produced by the first was consumed by the latter and the production of sulfide was boosted in the culture. As D. amilsii is not able to degrade glycerol, the co-culture represents a strategy to broaden the applicability of sulfur reduction at low pH with different sources of electron donors.

Microalgae are photosynthetic organisms that are found worldwide in many different aquatic environments and therefore display an immense biological diversity. They are a promising source of many useful polymers that have industrial applications such as food, fuel, material and pharmaceutical. One microalga that has gathered quite a research community is Botryococcus braunii. The reason for its scientific club is the fact it can synthetize long chain hydrocarbons molecules from C20 to C40. These hydrocarbons have been found in oil-shales and tests show that it can be used as aviation fuel. Besides producing hydrocarbons, some strains of B. braunii can produce exopolysaccharides (EPS) composed mainly of galactose and a small fraction of fucose. The EPS has interesting rheological properties for the food industry and potential active compounds that could be used in the pharmaceutical industry . Like many other microorganisms, microalgae in the natural environment are usually in the presence of bacteria. The presence of bacteria with microalgae can either have a beneficial or an antagonistic effect. For B. braunii little is known about the bacteria community present especially for the EPS producing strain. For that reason, the aim of this thesis was to investigate B. braunii’s associated bacteria with the aim of improving B. braunii’s biomass growth and hydrocarbon and EPS content. In chapter one, we introduced the topic of microalgae as a potential source of sustainable polymers and we introduced the species B. braunii, describing its characteristics and scientific interest. It is also introduced the topic of microalgae and bacteria associations by looking at other studies from literature. In chapter two, 16 publically available strains of B. braunii were ordered in culture banks and screened for biomass productivity, hydrocarbon and total carbohydrate content. The aim of the study was to identify one or more good strains that displayed high biomass productivity as well as hydrocarbon or total carbohydrate content. In seven strains out of 16 cultivated in 250 mL volume Erlenmeyer flaks, we detected 5 to 42 % content of hydrocarbons of the dry biomass with four strains producing botryococcenes (C30-C34) and three strains producing alkanes (C20-C25). Two strains showed high amounts of EPS content above 50 % per dry biomass. Seven strains comprising of the strains with higher biomass productivity plus the highest hydrocarbons and EPS content, were tested for scalability using bench scale 800 mL volume bubble column reactors. Two strains, AC761 which produces botryococcenes and CCALA778 which produces EPS, were selected as the most promising B. braunii strains for industrial production of hydrocarbon and EPS. In chapter three, we studied the bacterial community associated with B. braunii. We cultured 12 strains from the initial 16 from chapter 2 and extracted the DNA from samples taken over a time period of 12 days. It was clear from this study that B. braunii hosts a variety of bacterial species and still maintain its growth. The bacteria families Rhizobiaceae, Bradyrhizobiaceae and Comamonadaceae were found in all 12 strains. These families which belong to the phylum Proteobacteria could have an important role regarding B. braunii growth. Each strain displayed a different bacterial community composition but all the strains from the CAEN culture collection clustered near each other suggesting that the algae culture collection could have an influence on the bacterial community composition. Bacteria genus identification based on 16S rRNA gene amplicon similarity showed several genera present including Rhizobium spp. and Variovorax spp.. Two genera were found that are possibly linked to hydrocarbon degradation: Sphingomonas spp. and Rhodobacter spp.. In chapter four, we investigated further B. braunii CCALA778 which was shown in chapter 2 to accumulate high amounts of EPS. We investigated the effects of antibiotics on algal growth, EPS accumulation and bacterial community composition of CCALA778. Taxonomical identification by 16S rRNA gene analysis indicated that most of the bacteria present with CCALA778 were Gram-negative. Of all antibiotics and antibiotic mixes, only the treatment with Penicillin did not affect the growth of B. braunii. The remaining antibiotics halted the growth of CCALA778 while they were active. The exceptions were with the antibiotics Chloramphenicol, Gentamycin and Linezolid which permanently ceased the growth of CCALA778. The accumulation of EPS seemed to be related to biomass growth, but we did also observe a reduction of EPS with the cultures treated with Penicillin suggesting that bacteria could have an effect on the EPS content. Antibiotics had specific effects on the bacterial community with all treatments showing significant changes over time. The most efficient treatment in removing bacteria were the mixes Metronidazole-Rifampicin-Penicillin and Penicillin-Rifampicin which were the only treatments to show significant changes in the bacterial community when compared to the untreated cultures after 10 days of cultivation. Antibiotics and antibiotic mixes can create changes in the bacterial community but it is unlikely that they alone can lead to axenic B. braunii cultures. In chapter five, we used Ultra Violet-C light (UVC) to reduce bacteria diversity and abundance present in B. braunii CCALA778. UVC is highly effective in inactivating bacteria and for that reason is being investigated further in medicinal applications. After applying the UVC to B. braunii CCALA778, we were able to reduce the relative abundance of 16S rRNA genes assigned to bacteria to less than 1 % compared to the 70% in the non-treated cultures. With the UVC treated CCALA778 we observed several physiological changes. The UV treated cultures with reduced bacterial load showed nearly double the EPS accumulation when compared to the untreated. To confirm that we did not see an artefact in our results due to the UVC treatment, UVC treated cultures were also inoculated with bacteria from the untreated and we observed a reduction of EPS similar to what we saw with the untreated cultures. There were no changes to the EPS composition after the removal of the bacteria. Other physiological changes were observed, namely that colony size of B. braunii CCALA778 significantly increased when compared to the untreated culture and the UV treated with bacteria. We hypothesise that the increase in colony size was probably due to the fact there was more EPS accumulated which helped with cell aggregation. We also observed an increase on the biomass growth in the UV-treated CCALA778 which we hypothesized being related to the fact that there was none or hardly any competition for essential micronutrients such as phosphate. From this study we concluded that the associated bacteria present with B. braunii CCALA778 were antagonistic. We believe the reason why the bacteria were antagonistic is because of the readily available EPS which is a rich source of organic compounds that bacteria could use for their own proliferation allowing them to compete with B. braunii for essential nutrients. In chapter 6, we discuss the implications from our previous 4 experimental chapters. The aim of the study was to improve the biomass productivity and hydrocarbon and EPS content of the microalgae B. braunii. In brief, B. braunii displayed a wide range of physiological traits regarding biomass productivity and hydrocarbon and total carbohydrate content. We showed that B. braunii can co-habit with a wide range of bacteria diversity and abundance and that the associated bacteria were antagonistic to CCALA778 by affecting its biomass growth. We also showed that by removing the associated bacteria we can increase the EPS accumulation. Currently most of the research on microalgae and bacteria interactions, focus on the positive side, but we must understand also how bacteria can be antagonistic to microalga growth. Bacteria can be antagonistic to microalgae by competing for nutrients and also being detrimental to industrial process by degrading the product of interest in the case of organic carbons such as EPS. Therefore it is unlikely we can use the benefits that bacteria can provide such as enhancing growth to improve the cultivation of B. braunii and other similar microalgae species that secrete EPS. Since bacteria can be antagonistic to microalgae that secrete large amounts of organic compounds such as EPS, it is imperative to minimize contamination in large scale photobioreactors (PBR). It is important because in large scale PBR, contamination can occur leading to downtime of the reactors. If microalgae industry is to advance, it must develop PBR units that prevent contamination of bacteria from the surrounding environment.

The research of Bacterial Cell Factories aims to apply bacteria for production of biobased chemicals from renewable resources. The focus lies on thermophilic Gram-positives. This group of relatively unexplored thermophiles has many relevant characteristics that make them attractive as production organism for green chemicals. Development of genetic tools is a requirement for high-throughput engineering. The scientific challenge lies in exploring and exploiting the microbial physiology of the selected production organisms, involving an integrated approach of various disciplines. Successful development of such Bacterial Cell Factories is crucial for establishing the biobased economy.

Planten sterker maken waardoor ze beter bestand zijn tegen ziekten en plagen. Met dat doel worden biostimulanten of plantversterkers aan het wortelmilieu toegediend. Ze zijn sterk in opkomst, want ze vormen een alternatief voor chemische gewasbeschermingsmiddelen, waarvan het gebruik steeds meer onder druk komt te staan.

Elke zomer sterven grote hoeveelheden vogels en vissen tijdens aanhoudend warm weer.

Porous aluminum oxide (PAO) is a nanostructured material used for various biotechnological applications, including the culturing microorganisms and other types of cells. The ability to chemically modify the PAO surface and tailor its surface properties is a promising way to expand and refine its applications. The immobilization of biomolecules on PAO that specifically interact with and bind to target bacteria would enable the capture and subsequent growth of bacteria on the same surface, and this was the ultimate goal of the research presented in this thesis. After a general introduction to the overall subject of this thesis, presented in Chapter 1, the most commonly used and recent methods to prepare glycosurfaces are reviewed and compared on their merits and drawbacks in Chapter 2. Although there are a great number of techniques, the main challenge that still remains is to develop an accessible, reproducible and inexpensive approach that produces well-defined and stable glycosurfaces using as few steps as possible. The most used analytical techniques for the characterization of glycosurfaces and several applications of these surfaces in the binding, capture, and sensing of bacteria and bacterial toxins were also discussed in Chapter 2. Biofunctionalization of surfaces in general requires a stepwise approach, in which it is very important to have a stable monolayer as the first step. At the beginning of this research it was known that various functional groups were able to react with (porous) aluminum oxide, but there was no comprehensive study comparing the stability of these modified surfaces under the conditions that are important for microbiological applications. In Chapter 3, the PAO surface was modified with various functional groups known to react with PAO (carboxylic acid, α-hydroxycarboxylic acid, alkyne, alkene, phosphonic acid, and silane), and the stability of these modified surfaces was assessed over a range of pH and temperatures that are relevant for microbial growth. Silane and phosphonate-modified PAO surfaces with a hydrophobic monolayer proved to be the most stable ones, but the phosphonate modification was both more easily applied and reproducible. This modification was stable for at least two weeks in buffer solutions with pH values between 4 and 8, and at temperatures up to 40 °C. Only at elevated temperatures of 60 °C and 80 °C under hydrolytic conditions it was observed that the stability of the same monolayer on PAO decreased gradually. As a proof-of-principle for the biofunctionalization and bacterial capture on this PAO phosphonate monolayer, an alkyne-terminated monolayer was biofunctionalized via a CuAAC click reaction with an azido-mannoside and the binding and growth of Lactobacillus plantarum was successfully demonstrated. In Chapter 4 various approaches to install reactive groups onto the phosphonate-modified PAO surface were developed, creating a (bio)functionalization “tool-box”. PAO surfaces presenting different terminal reactive groups were prepared, such as azide, alkyne, alkene, thiol, isothiocyanate, and N-hydroxysuccinimide (NHS), starting from a single, straightforward and stable initial modification with a bromo-terminated phosphonic acid. These reactive surfaces were then used to immobilize (bio)molecules, including carbohydrates and proteins. Fluorescently labeled bovine serum albumin (BSA) was covalently immobilized on the PAO surface as a proof-of-principle, and it was shown that a range of bacteria could still grow on the BSA-functionalized PAO surface. With a PAO (bio)functionalization tool-box in hand, the successful proof-of-principle mannoside-dependent binding and growth of L. plantarum on PAO (Chapter 3) was further investigated and expanded upon (Chapter 5). The parameters involved in the preparation of these surfaces and in the binding with L. plantarum were investigated in more detail in Chapter 5, such as the nature of the spacer connected to the mannoside derivative and the presence of soluble carbohydrates and bovine serum albumin (BSA) in the medium. The surfaces with the azido-mannoside with the long hydrophobic spacer showed the best binding of L. plantarum when compared to a long PEG-based hydrophilic spacer and a short hydrophobic one. The presence of a soluble a-glucoside did not prevent the binding of the bacteria to the mannose-presenting PAO, and similar results were obtained when BSA was present. Additionally, a mutant strain of L. plantarum that does not have the mannose-specific adhesion was not able to bind to the mannose-presenting PAO. When taken together, this proves that the mannoside–adhesin interaction is the main mechanism of binding the bacteria to the mannose-biofunctionalized PAO in this system. In Chapter 6, the NHS-terminated PAO developed in Chapter 4 was used for the immobilization of antibodies against Escherichia coli. After an extensive optimization of the modification chemistry of the surfaces and the incubation conditions, commercially available anti-E. coli antibodies were immobilized on the PAO surface. Binding and washing experiments indeed demonstrated increased binding of E. coli on the antibody-presenting PAO surfaces, providing avenues for testing other bacteria such as Lactobacillus rhamnosus GG widely used in probiotic formulations worldwide. In Chapter 7, the most important achievements of this project are discussed, together with additional ideas and recommendations for further research. Most notably some preliminary results are presented on the immobilization of two antibodies against L. rhamnosus GG: anti-L. rhamnosus GG, against the whole bacterial cell, and anti-SpaC, against only the SpaC part of the pili present on the cell surface of L. rhamnosus GG. Anti-L. rhamnosus GG antibody showed promising but not yet optimal increased binding of L. rhamnosus GG. Finally, some reflections on PAO and its (bio)functionalization are provided in the context of a risk analysis and technology assessment.

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.

Fibrillar structures are important structuring elements for food products. Understanding the behaviour of fibrillar structures in complex food systems is essential for successful industrial applications. This thesis presents the behaviour of two different fibrillar structures, i.e. whey protein isolate (WPI) fibrils and bacterial cellulose (BC) microfibrils in mixtures under various conditions. The WPI fibrils are prepared from WPI and the BC microfibrils are extracted from commercial available ‘Nata de Coco’ by high-energy de-agglomeration. In Chapter 1, a general introduction is given, where we introduce two different fibrillar structures that were studied in this thesis. Also, the aim and the outline of the thesis are presented. In Chapter 2, 3, 4 and 5, the behaviour of mixtures containing WPI and BC microfibrils under different conditions are investigated. By varying the concentration ratios, pH, NaCl concentration and further applying heating treatment, their physico-chemical properties in mixed solutions, mixed solutions after heating and further heat-induced mixed gels are investigated and characterized at both pH 2 and pH 7. In general, both mixing WPI and BC microfibrils without heating and subsequently applying heating treatment lead to stable and homogeneous mixtures at pH 7, as long as BC microfibril concentration is above a critical value. Microscopic images showed that the WPI aggregates and BC microfibrils co-existed in the system. WPI denatured and aggregated in the mixture in the same way as when it is heated alone. Upon gelation, the WPI and BC microfibrils form a duplex gel consisting of two independent and homogeneous networks spanning the whole system. At pH 2, the WPI and BC microfibrils also form stable and homogeneous mixtures in the liquid state, both before and after heating. Microscopic images showed two fibrillar structures that are uniformly and independently present. Upon gelation at higher WPI concentration, a bi-fibrillar gel is formed consisting of a WPI fibrilllar gel and BC microfibrillar gel that co-exist. In Chapter 6 and 7, the behaviour of WPI fibrils at pH 2 in dispersions containing spheres, i.e. emulsions and polystyrene latex dispersions are studied. When WPI and spheres are both positively charged (i.e. WPI-stabilized emulsion), we observed depletion flocculation and depletion stabilization when the WPI fibril concentration increases. When WPI and the spheres are oppositely charged (i.e. polystyrene latex dispersions), bridging flocculation and steric/electrostatic stabilization were observed at low WPI fibril concentration, followed by depletion flocculation and depletion stabilization upon increasing WPI fibril concentrations. In Chapter 8 the stability of emulsions at pH 2 in the presence of only BC microfibrils and in the presence of both BC microfibrils and WPI fibrils was studied. When only BC microfibrils added at a sufficiently high concentration, the emulsions are stabilized by the presence of a yield stress as generated by the BC network. When both WPI fibrils and BC microfibrils are added to the emulsions, the networks they form behave in the same way, as when they are added to the emulsions separately. The WPI fibrils induced depletion flocculation and stabilization of the emulsions, despite the presence of the BC microfibrils. However, at high enough BC microfibril concentrations, the emulsions can be stabilized against depletion flocculation as induced by the WPI fibrils. The competition between stabilization and/or de-stabilization induced by the BC microfibrils and the WPI fibrils can lead to emulsions with different microstructures and rheological properties. A general discussion on the results obtained in this thesis is presented in Chapter 9, which includes recommendations for further research and concluding remarks.

Huijbers, P.M.C. (2016). Transmission of antibiotic resistance from animals to humans: Broilers as a reservoir of ESBL-producing bacteria. PhD thesis, Wageningen University, Wageningen, the Netherlands. Antibiotic resistance in animals becomes a public health issue when there is transmission of antibiotic resistant bacteria, or their resistance genes, from animals to humans. β-lactam antibiotics are critically important for the treatment of human bacterial infections. Resistance to this class of antibiotics, mediated by extended-spectrum β-lactamases (ESBL) has emerged. Broilers might contribute to transmission to humans due to the high prevalence of ESBL-producing Enterobacteriaceae among their intestinal biome, compared to other livestock species, companion animals, and wildlife. Transmission to humans might occur via the food chain, by direct contact or via the environment. The aim was to investigate transmission of antibiotic resistant bacteria between animals and humans, and more specifically transmission of ESBL-producing E. coli between broilers, and between broilers and humans in varying degrees of contact with these animals. Systematically collected and categorised evidence from literature showed that clinically relevant antibiotic resistant bacteria were present in the natural environment, that is in soil, water, air and wildlife. It was therefore hypothesised that humans in areas with high broiler densities might have an increased risk for carriage of ESBL-producing Enterobacteriaceae. This hypothesis was rejected, as the observed risk was lower for these individuals. The situation might be different for individuals living on broiler farms as ESBL-producing E. coli were detected on all investigated farms. Among broilers, the within farm prevalence approached 100%, and there was no difference between conventional and organic farms at five weeks, i.e. just before slaughter on conventional farms. On organic farms, the prevalence decreased to 80.0% at 70 days, i.e. slaughter age. Not only transmission to humans via the farm environment, but close physical contact with broilers might, therefore, lead to increased risk for carriage. Prevalence among farmers, their family members and employees on both conventional (19.1%) and organic (18.5%) broiler farms was higher compared to humans in the general population (5.1%). Moreover, people in close contact with live broilers showed the highest risk (27.1 vs. 14.3%). Evidence for clonal transmission of ESBL-producing E. coli between humans and broilers was found on conventional farms, and horizontal gene transfer was suspected on both conventional and organic farms. Even without selection pressure from antibiotics ESBL-producing E. coli were able to transmit and persist in an organic broiler flock, which shows that broilers form a reservoir of antibiotic resistance genes. This leads to an increased risk of carriage of humans on farms through direct contact with broilers and possibly via the direct farm environment. As only a very small percentage of the general population is exposed to live broilers, direct contact with broilers does not appear to be important for carriage in the general human population.

Organohalide-respiring bacteria (OHRB) are able to breathe natural and anthropogenically produced organohalides persistent in a broad range of oxygen-depleted environments. Therefore, these microorganisms are of high interest for organohalide-contaminated site bioremediation and natural halogen and carbon cycle. Nevertheless, to assess and adjust in situ bioremediation strategies and to enhance current understanding about the role of OHRB in natural habitats, thorough understanding of their ecophysiology and interaction with surrounding biotic and abiotic forces is necessary. To this end, this thesis focused on exploring ecophysiology and environmental distribution of OHRB in pristine and contaminated sites and unraveling their interactions with the co-existing microbial guilds in the community and geochemical parameters by application of a suite of physiological, molecular and geochemical analyses. Based on a comprehensive overview of currently known organohalide-respiring isolates and their environmental distribution, the presence of yet unknown OHRB in extreme environments was proposed as the known organohalide-respiring isolates survive/thrive at a moderate range of pH and salinity in laboratory culture. Therefore, the OHRB were surveyed in alkaline and hypersaline sediments collected from Lake Strawbridge, Western Australia, that was known to emit organohalides. As a result, for the first time, the dechlorination of chloroform and perchloroethene (PCE) to dichloromethane and trichloroethene, respectively， was documented from an alkaline hypersaline pristine environment. Corrinoids are essential cofactors for the activity of reductive dehalogenase enzymes. Ironically, some OHRB are reported to be corrinoid auxotrophs. Using transcriptional analysis and shotgun proteomics, here we show corrinoid auxotrophy in Dehalobacter restrictus PER-K23T. This detrimental deficiency seems to be compensated by up-regulation of relevant cobalamin salvaging and transport pathways to ensure sufficient corrinoid supply under partial corrinoid starvation. Hence, such OHRB incapable of de novo corrinoid synthesis will be dependent on non-dechlorinating community members to fulfill their nutritional needs indicating paramount importance of syntrophic interactions in supporting robust growth and activity of OHRB. Bacterial community analysis of chlorinated benzene dechlorinating consortia derived from contaminated harbour sludge suggested members of the Bacteroidetes phylum and Clostridiales order as well as sulfate-reducing Deltaproteobacteria as putative stimulating guilds that provide electron donor and/or organic cofactors to OHRB i.e. D. mccartyi and Dehalobacter. However, despite well-controlled lab condition, syntrophic interactions could be influenced by geochemical parameters under field settings. Accordingly, analysis of geochemical and microbial determinants of OHR at a site biostimulated by glycerol injection further verified supportive role of fermenters and sulfate reducers under highly reduced condition following biostimulation. However, towards the end of field experiment, reducing condition faded and sulfate increased concurrent with the appearance of Epsilonproteobacteria and Deferribacteres as putative oxidizers of reduced sulfur compounds. The latter guilds might serve as detoxifiers of sulfide and thereby stimulate D. mccartyi, but could also be inhibitory as successors of the more important syntrophic fermenting and sulfate reducing bacteria. In conclusion, this thesis expands our understanding of ecophysiology and environmental distribution of OHRB, addressing their presence in pristine environments as well as providing further evidence for their dependencies on other microbial community members in order to meet their nutritional requirements. Hence, research described here strengthens the scientific foundation for evaluating and optimizing strategies for the bioremediation of organohalide-contaminated sites and expands the natural niche of OHRB to extreme pristine environments.

Huijbers, P.M.C. (2016). Transmission of antibiotic resistance from animals to humans: Broilers as a reservoir of ESBL-producing bacteria. PhD thesis, Wageningen University, Wageningen, the Netherlands. Antibiotic resistance in animals becomes a public health issue when there is transmission of antibiotic resistant bacteria, or their resistance genes, from animals to humans. β-lactam antibiotics are critically important for the treatment of human bacterial infections. Resistance to this class of antibiotics, mediated by extended-spectrum β-lactamases (ESBL) has emerged. Broilers might contribute to transmission to humans due to the high prevalence of ESBL-producing Enterobacteriaceae among their intestinal biome, compared to other livestock species, companion animals, and wildlife. Transmission to humans might occur via the food chain, by direct contact or via the environment. The aim was to investigate transmission of antibiotic resistant bacteria between animals and humans, and more specifically transmission of ESBL-producing E. coli between broilers, and between broilers and humans in varying degrees of contact with these animals. Systematically collected and categorised evidence from literature showed that clinically relevant antibiotic resistant bacteria were present in the natural environment, that is in soil, water, air and wildlife. It was therefore hypothesised that humans in areas with high broiler densities might have an increased risk for carriage of ESBL-producing Enterobacteriaceae. This hypothesis was rejected, as the observed risk was lower for these individuals. The situation might be different for individuals living on broiler farms as ESBL-producing E. coli were detected on all investigated farms. Among broilers, the within farm prevalence approached 100%, and there was no difference between conventional and organic farms at five weeks, i.e. just before slaughter on conventional farms. On organic farms, the prevalence decreased to 80.0% at 70 days, i.e. slaughter age. Not only transmission to humans via the farm environment, but close physical contact with broilers might, therefore, lead to increased risk for carriage. Prevalence among farmers, their family members and employees on both conventional (19.1%) and organic (18.5%) broiler farms was higher compared to humans in the general population (5.1%). Moreover, people in close contact with live broilers showed the highest risk (27.1 vs. 14.3%). Evidence for clonal transmission of ESBL-producing E. coli between humans and broilers was found on conventional farms, and horizontal gene transfer was suspected on both conventional and organic farms. Even without selection pressure from antibiotics ESBL-producing E. coli were able to transmit and persist in an organic broiler flock, which shows that broilers form a reservoir of antibiotic resistance genes. This leads to an increased risk of carriage of humans on farms through direct contact with broilers and possibly via the direct farm environment. As only a very small percentage of the general population is exposed to live broilers, direct contact with broilers does not appear to be important for carriage in the general human population.

Op basis van internationaal literatuuronderzoek blijken er interessante kansen voor toepassing van microalgen producten in landbouwgewassen te zijn. In dit rapport worden zowel groene algen als cyanobacteriën gedefinieerd als microalgen. Stoffen uit microalgen (met name uit cyanobacteriën) blijken in diverse onderzoeken uit de literatuur een goede bestrijding te geven van verschillende schimmels en aaltjes die in landbouwgewassen schade aanrichten. Verder is er (buiten de landbouw) insecticide-, molluscicide-, herbicide- en algacidewerking met stoffen uit cyanobacteriën aangetoond. In Nederland zijn zeewier- en algenextracten krachtens de Verordening gewasbescherming als werkzame stof goedgekeurd voor de groeiregulatie van planten. Het gebruik als bodemverbeteraar of als plantenstimulator is vooralsnog veel minder gereguleerd dan als gewasbeschermingsmiddel. Om de kansen met microalgen te benutten zou er verder geïnvesteerd moeten worden in onderzoek. Het zou duidelijk moeten worden welke werkzame stoffen uit micro algen of welke algenpreparaten andere organismen bestrijden, in welke formulering en met welke dosering. De bestrijdende, bemestende, bodem verbeterende, plantweerstand verhogende en milieueffecten van algentoepassingen zouden vergeleken moeten worden met conventionele methoden.

Op basis van internationaal literatuuronderzoek blijken er interessante kansen voor toepassing van microalgen producten in landbouwgewassen te zijn. In dit rapport worden zowel groene algen als cyanobacteriën gedefinieerd als microalgen. Stoffen uit microalgen (met name uit cyanobacteriën) blijken in diverse onderzoeken uit de literatuur een goede bestrijding te geven van verschillende schimmels en aaltjes die in landbouwgewassen schade aanrichten. Verder is er (buiten de landbouw) insecticide-, molluscicide-, herbicide- en algacidewerking met stoffen uit cyanobacteriën aangetoond. In Nederland zijn zeewier- en algenextracten krachtens de Verordening gewasbescherming als werkzame stof goedgekeurd voor de groeiregulatie van planten. Het gebruik als bodemverbeteraar of als plantenstimulator is vooralsnog veel minder gereguleerd dan als gewasbeschermingsmiddel. Om de kansen met microalgen te benutten zou er verder geïnvesteerd moeten worden in onderzoek. Het zou duidelijk moeten worden welke werkzame stoffen uit micro algen of welke algenpreparaten andere organismen bestrijden, in welke formulering en met welke dosering. De bestrijdende, bemestende, bodem verbeterende, plantweerstand verhogende en milieueffecten van algentoepassingen zouden vergeleken moeten worden met conventionele methoden.

Porous aluminum oxide (PAO) is a nanostructured material used for various biotechnological applications, including the culturing microorganisms and other types of cells. The ability to chemically modify the PAO surface and tailor its surface properties is a promising way to expand and refine its applications. The immobilization of biomolecules on PAO that specifically interact with and bind to target bacteria would enable the capture and subsequent growth of bacteria on the same surface, and this was the ultimate goal of the research presented in this thesis. After a general introduction to the overall subject of this thesis, presented in Chapter 1, the most commonly used and recent methods to prepare glycosurfaces are reviewed and compared on their merits and drawbacks in Chapter 2. Although there are a great number of techniques, the main challenge that still remains is to develop an accessible, reproducible and inexpensive approach that produces well-defined and stable glycosurfaces using as few steps as possible. The most used analytical techniques for the characterization of glycosurfaces and several applications of these surfaces in the binding, capture, and sensing of bacteria and bacterial toxins were also discussed in Chapter 2. Biofunctionalization of surfaces in general requires a stepwise approach, in which it is very important to have a stable monolayer as the first step. At the beginning of this research it was known that various functional groups were able to react with (porous) aluminum oxide, but there was no comprehensive study comparing the stability of these modified surfaces under the conditions that are important for microbiological applications. In Chapter 3, the PAO surface was modified with various functional groups known to react with PAO (carboxylic acid, α-hydroxycarboxylic acid, alkyne, alkene, phosphonic acid, and silane), and the stability of these modified surfaces was assessed over a range of pH and temperatures that are relevant for microbial growth. Silane and phosphonate-modified PAO surfaces with a hydrophobic monolayer proved to be the most stable ones, but the phosphonate modification was both more easily applied and reproducible. This modification was stable for at least two weeks in buffer solutions with pH values between 4 and 8, and at temperatures up to 40 °C. Only at elevated temperatures of 60 °C and 80 °C under hydrolytic conditions it was observed that the stability of the same monolayer on PAO decreased gradually. As a proof-of-principle for the biofunctionalization and bacterial capture on this PAO phosphonate monolayer, an alkyne-terminated monolayer was biofunctionalized via a CuAAC click reaction with an azido-mannoside and the binding and growth of Lactobacillus plantarum was successfully demonstrated. In Chapter 4 various approaches to install reactive groups onto the phosphonate-modified PAO surface were developed, creating a (bio)functionalization “tool-box”. PAO surfaces presenting different terminal reactive groups were prepared, such as azide, alkyne, alkene, thiol, isothiocyanate, and N-hydroxysuccinimide (NHS), starting from a single, straightforward and stable initial modification with a bromo-terminated phosphonic acid. These reactive surfaces were then used to immobilize (bio)molecules, including carbohydrates and proteins. Fluorescently labeled bovine serum albumin (BSA) was covalently immobilized on the PAO surface as a proof-of-principle, and it was shown that a range of bacteria could still grow on the BSA-functionalized PAO surface. With a PAO (bio)functionalization tool-box in hand, the successful proof-of-principle mannoside-dependent binding and growth of L. plantarum on PAO (Chapter 3) was further investigated and expanded upon (Chapter 5). The parameters involved in the preparation of these surfaces and in the binding with L. plantarum were investigated in more detail in Chapter 5, such as the nature of the spacer connected to the mannoside derivative and the presence of soluble carbohydrates and bovine serum albumin (BSA) in the medium. The surfaces with the azido-mannoside with the long hydrophobic spacer showed the best binding of L. plantarum when compared to a long PEG-based hydrophilic spacer and a short hydrophobic one. The presence of a soluble a-glucoside did not prevent the binding of the bacteria to the mannose-presenting PAO, and similar results were obtained when BSA was present. Additionally, a mutant strain of L. plantarum that does not have the mannose-specific adhesion was not able to bind to the mannose-presenting PAO. When taken together, this proves that the mannoside–adhesin interaction is the main mechanism of binding the bacteria to the mannose-biofunctionalized PAO in this system. In Chapter 6, the NHS-terminated PAO developed in Chapter 4 was used for the immobilization of antibodies against Escherichia coli. After an extensive optimization of the modification chemistry of the surfaces and the incubation conditions, commercially available anti-E. coli antibodies were immobilized on the PAO surface. Binding and washing experiments indeed demonstrated increased binding of E. coli on the antibody-presenting PAO surfaces, providing avenues for testing other bacteria such as Lactobacillus rhamnosus GG widely used in probiotic formulations worldwide. In Chapter 7, the most important achievements of this project are discussed, together with additional ideas and recommendations for further research. Most notably some preliminary results are presented on the immobilization of two antibodies against L. rhamnosus GG: anti-L. rhamnosus GG, against the whole bacterial cell, and anti-SpaC, against only the SpaC part of the pili present on the cell surface of L. rhamnosus GG. Anti-L. rhamnosus GG antibody showed promising but not yet optimal increased binding of L. rhamnosus GG. Finally, some reflections on PAO and its (bio)functionalization are provided in the context of a risk analysis and technology assessment.

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.

Voor veel sportvissers is karper de favoriete vissoort. Dankzij een doordacht water- en visstandbeheer kent ons land een gezonde karperpopulatie. Tocht treedt er af en toe sterfte op bij deze vissen. Mede op verzoek van Sportvisserij Nederland onderzoekt het Centraal Veterinair Instituut in Lelystad de oorzaak van deze sterftes.

Recently discovered microorganisms affiliated to the bacterial phylum NC10, named “Candidatus Methylomirabilis oxyfera”, perform nitrite-dependent anaerobic methane oxidation. These microorganisms could be important players in a novel way of anaerobic wastewater treatment where ammonium and residual dissolved methane might be removed at the expense of nitrate or nitrite. To find suitable inocula for reactor startup, ten selected wastewater treatment plants (WWTPs) located in The Netherlands were screened for the endogenous presence of M. oxyfera using molecular diagnostic methods. We could identify NC10 bacteria with 98% similarity to M. oxyfera in nine out of ten WWTPs tested. Sludge from one selected WWTP was used to start a new enrichment culture of NC10 bacteria. This enrichment was monitored using specific pmoA primers and M. oxyfera cells were visualized with fluorescence oligonucleotide probes. After 112 days, the enrichment consumed up to 0.4 mM NO2− per day. The results of this study show that appropriate sources of biomass, enrichment strategies, and diagnostic tools existed to start and monitor pilot scale tests for the implementation of nitrite-dependent methane oxidation in wastewater treatment at ambient temperature. Electronic supplementary material The online version of this article (doi:10.1007/s00253-011-3361-9) contains supplementary material, which is available to authorized users.

Per 1 februari 2009 ging het project PROMICROBE van start. Het project richt zich op de invloed van bacteriën op de ontwikkeling, groei, productie en overleving van vissen in teeltsystemen. Een vergelijking wordt gemaakt tussen tilapia, kabeljauw en zeebaars. Het einddoel van het onderzoek is het bedrijfszeker kweken van gezonde vissen in duurzame en stabiele teeltystemen

Wageningen UR gaat de komende jaren investeren in synthetische biologie. Het is benoemd als een van de investeringsthema’s in het strategisch plan. Een jong vakgebied, waarbij onderzoekers bacteriën verbouwen en ontwerpen voor industriële toepassingen. Hoogleraar Martins dos Santos, die onlangs twee grote EU-projecten binnenhaalde, gaat een actieplan schrijven.

In this study the diurnal variations in bacteria and endotoxin concentrations and emissions in houses for finishing pigs and broilers were determined.

In this study the diurnal variations in bacteria and endotoxin concentrations and emissions in houses for finishing pigs and broilers were determined. De doelstelling van dit onderzoek is om de binnen-dag variatie in bacterie- en endotoxineconcentraties en –emissies vast te stellen. Dit is gedaan voor twee belangrijke diercategorieën, namelijk vleesvarkens en vleeskuikens.

HYVOLUTION is the acronym of an Integrated Project ¿Non-thermal production of pure hydrogen from biomass¿ which has been granted in the Sixth EU Framework Programme on Research, Technological Development and Demonstration, Priority 6.1.ii, Sustainable Energy Systems. The aim of HYVOLUTION: ¿Development of a blue-print for an industrial bioprocess for decentral hydrogen production from locally produced biomass¿ adds to the number and diversity of H2 production routes giving greater security of supply at the local and regional level. Moreover, this IP contributes a complementary strategy to fulfil the increased demand for renewable hydrogen expected in the transition to the Hydrogen Economy. The novel approach in HYVOLUTION is based on a combined bioprocess employing thermophilic and phototrophic bacteria, to provide the highest hydrogen production efficiency in small-scale, cost-effective industries. In HYVOLUTION, 11 EU countries, Turkey and Russia are represented to assemble the critical mass needed to make a breakthrough in cost-effectiveness.

Veel paardekastanjes in Nederland lijden aan de enstige bloedingsziekte, hierover is een landelijke inventarisatie gehouden. Het artikel behandelt wat tegen deze ziekte gedaan wordt

Nederland is een water- en visrijk land. In onze binnenwaterenkomen 45 inheemse en 14 uitheemse vissoortenvoor. Hoewel de waterkwaliteit en de leefomgeving vanvissen sterk zijn verbeterd, kunnen vissen ziek worden.Welke ziekten zijn dat? En welke nieuwe visziektenvormen een risico voor onze vissen? Het CIDC-Lelystaddoet al bijna 21 jaar onderzoek aan visziekten. Olga Haenenen Marc Engelsma gaan in op de meest voorkomendevisziekten bij zowel wilde als gekweekte vis.