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Accumulation of non-degradable plastic waste in the environment might be prevented by the use of biodegradable polyhydroxyalkanoate (PHA). In this study, the thermophile Schlegelella thermodepolymerans produced up to 80 wt% PHA based on dry cell mass. The largest PHA granules were found in the cells within 48 h using 20 g/L xylose, a C/N ratio of 100, an initial pH of 7, at 50 °C. The substrate consumption, pH changes, and cell growth were monitored, revealing the time dependency of PHA production in S. thermodepolymerans. The metabolic pathways from xylose to PHA were identified based on proteomic analysis, revealing involvement of classic phaCAB, de novo fatty acid biosynthesis, and fatty acid β-oxidation. In addition, it was shown that S. thermodepolymerans degraded extracellular PHA with a high efficiency at 50 °C.

The bacterium Escherichia coli initiates replication once per cell cycle at a precise volume per origin and adds an on average constant volume between successive initiation events, independent of the initiation size. Yet, a molecular model that can explain these observations has been lacking. Experiments indicate that E. coli controls replication initiation via titration and activation of the initiator protein DnaA. In chapters 2, 3 and 4, we study by mathematical modelling how these two mechanisms interact to generate robust replication-initiation cycles. We first show that a mechanism solely based on titration generates stable replication cycles at low growth rates, but inevitably causes premature reinitiation events at higher growth rates. In this regime, the DnaA activation switch becomes essential for stable replication initiation. Conversely, while the activation switch alone yields robust rhythms at high growth rates, titration can strongly enhance the stability of the switch at low growth rates. Our analysis thus predicts that both mechanisms together drive robust replication cycles at all growth rates. In addition, it reveals how an origin-density sensor yields adder correlations. Initiating replication synchronously at multiple origins of replication allows the bacterium E. coli to divide even faster than the time it takes to replicate the entire chromosome in nutrient rich environments. What mechanisms give rise to synchronous replication initiation remains however poorly understood. In chapter 5, we identify four distinct synchronization regimes depending on two quantities: the duration of the so-called licensing period during which the initiation potential in the cell remains high after the first origin has fired and the duration of the blocking period during which already initiated origins remain blocked. For synchronous replication initiation, the licensing period must be long enough such that all origins can be initiated, but shorter than the blocking period to prevent reinitiation of origins that have already fired. We find an analytical expression for the degree of synchrony as a function of the duration of the licensing period, which we confirm by simulations. Our model reveals that the delay between the firing of the first and the last origin scales with the coefficient of variation (CV) of the initiation volume. Matching these to the values measured experimentally shows that the firing rate must rise with the cell volume with an effective Hill coefficient that is at least 20; the probability that all origins fire before the blocking period is over is then at least 94%. Our analysis thus reveals that the low CV of the initiation volume is a consequence of synchronous replication initiation. Finally, we show that the previously presented molecular model for the regulation of replication initiation in E. coli can give rise to synchronous replication initiation for biologically realistic parameters. Recent developments in synthetic biology may bring the bottom-up generation of a synthetic cell within reach. A key feature of a living synthetic cell is a functional cell cycle, in which DNA replication and segregation as well as cell growth and division are well integrated. In chapter 6, we describe different approaches to recreate these processes in a synthetic cell, based on natural systems and/or synthetic alternatives. Although some individual machineries have recently been established, their integration and control in a synthetic cell cycle remain to be addressed. In this chapter, we discuss potential paths towards an integrated synthetic cell cycle.

Millions of microorganisms inhabit the soil, and some of them contribute to the growth of plants. To gain a better understanding of how microbes promote plant growth, we compared the impact of fungal and bacterial communities on litter decomposition and plant growth. Our study revealed that fungi are the primary decomposers of plant litter and have a more significant impact on nutrient cycling than bacteria. Furthermore, when the fungal and bacterial communities are working together, they have a complementary effect on plant growth that is greater than when either community is working alone. Arbuscular mycorrhizal fungi (AMF) are recognized as one of the most important types of plant symbiotic fungi. AMF are widely distributed in the soil and colonize plant roots. They can functionally extend the plant root system and reach areas in the soil beyond the plant roots, enabling plants to access more water and nutrients. We investigated the microbial community that surrounds the AMF hyphae and discovered that fungal hyphae are colonized by specific bacteria and protists. We demonstrate that the microbes that colonize the fungal hyphae play a crucial role in facilitating mycorrhizal development and assisting plant nutrient uptake. We showed that a particular bacterium of the genus Devosia is associated with AMF hyphae, interacts synergistically with the mycorrhiza and promotes plant growth by facilitating nitrogen uptake. This thesis deepens our understanding of the interactions between different microbial groups and their roles in litter decomposition and mycorrhization processes. A better understanding of plant-microbe interactions is essential for the development of effective application strategies for plant-growth-promoting microbes in agriculture. Ultimately microbes can help create sustainable agricultural practices that rely less on chemical inputs for food production.

De lectorale rede gehouden door Janneke Krooneman tijdens haar lectorinstallatie op 5 april.

The application of micro-organisms has been increasingly suggested as sustainable agricultural approach. Soil micro-organisms include bacteria, archaea, fungi and protists. The majority of soil protists are free-living predators that feed, among others, on bacteria. Through their predatory activity, they typically unlock nutrients that can benefit plants. In addition, because soil protists do not equally feed on all bacteria, they influence their prey community structure which can further lead to an enrichment in plant-beneficial taxa. One challenge but also an opportunity of soil protist ecology lies in the high phylogenetic diversity of protists that likely mirrors an equally high functionality: each protist species could have a distinct impact on plant development. Most of the current understanding is, however, based on few model species and successful protist application typically follows a trial and error methodology with little a priori knowledges. In the present work, we investigated the potential of phylogenetically diverse soil protists to support plant development, especially via their impact on the bacterial community composition. We first pursued to identify relevant protist and bacterial traits to better understand and predict predator-prey interactions. We were able to relate the ability of bacteria to inhibit their predators to their ability to suppress pathogens (Chapter 2). We identified for each of the protist tested distinct prey consumption patterns which were related to their predatory impacts on soil bacterial communities: protist species that could feed on the same set of bacteria in a plate assay had a similar impact on the bacterial community structure in the soil (Chapter 3). After investigating the predator-prey interactions, we examined different aspects of protist application to best support plant development by using lettuce, Lactuca sativa, as model plant. We found that the effect on the plant varied depending on the protist species inoculated (Chapter 4) and that application of protists before transferring the plant into the soil led to the best yield (Chapter 5). In chapter 6, I synthesized the obtained results, further examined the context-dependency of the predatory impact of protists and suggested plate assays to be promising in soil protist ecology. The results of this work highlight that protist predatory impact is taxon specific, both on its prey community and on plant development. Further, effects on plant development are not necessarily beneficial or related to an overall change of the bacterial community composition. We conclude that predatory protists are an essential component of the soil microbiome functioning and have the potential to support plant development.

The quantification and identification of new plasmid-acquiring bacteria in representative mating conditions is critical to characterize the risk of horizontal gene transfer in the environment. This study aimed to quantify conjugation events resulting from manure application to soils and identify the transconjugants resulting from these events. Conjugation was quantified at multiple time points by plating and flow cytometry, and the transconjugants were recovered by fluorescence-activated cell sorting and identified by 16S rRNA sequencing. Overall, transconjugants were only observed within the first 4 days after manure application and at values close to the detection limits of this experimental system (1.00-2.49 log CFU/g of manured soil, ranging between 10(-5) and 10(-4) transconjugants-to-donor ratios). In the pool of recovered transconjugants, we found amplicon sequence variants (ASVs) of genera whose origin was traced to soils (Bacillus and Nocardioides) and manure (Comamonas and Rahnella). This work showed that gene transfer from fecal to soil bacteria occurred despite the less-than-optimal conditions faced by manure bacteria when transferred to soils, but these events were rare, mainly happened shortly after manure application, and the plasmid did not colonize the soil community. This study provides important information to determine the risks of AMR spread via manure application.

Application of animal manure to soils results in the introduction of manure-derived bacteria and their antimicrobial resistance genes (ARGs) into soils. ResCap is a novel targeted-metagenomic approach that allows the detection of minority components of the resistome gene pool without the cost-prohibitive coverage depths and can provide a valuable tool to study the spread of antimicrobial resistance (AMR) in the environment. We used high-throughput sequencing and qPCR for 16S rRNA gene fragments as well as ResCap to explore the dynamics of bacteria, and ARGs introduced to soils and adjacent water ditches, both at community and individual scale, over a period of three weeks. The soil bacteriome and resistome showed strong resilience to the input of manure, as manuring did not impact the overall structure of the bacteriome, and its effects on the resistome were transient. Initially, manure application resulted in a substantial increase of ARGs in soils and adjacent waters, while not affecting the overall bacterial community composition. Still, specific families increased after manure application, either through the input of manure (e.g., Dysgonomonadaceae) or through enrichment after manuring (e.g., Pseudomonadaceae). Depending on the type of ARG, manure application resulted mostly in an increase (e.g., aph(6)-Id), but occasionally also in a decrease (e.g., dfrB3) of the absolute abundance of ARG clusters (FPKM/kg or L). This study shows that the structures of the bacteriome and resistome are shaped by different factors, where the bacterial community composition could not explain the changes in ARG diversity or abundances. Also, it highlights the potential of applying targeted metagenomic techniques, such as ResCap, to study the fate of AMR in the environment.

De samenstelling van de bacterie- en schimmelgemeenschap als geheel is specifiek voor elk onderdeel van de kringloop op een melkveebedrijf. Dat laat een verkennend onderzoek gefinancierd door de Triodos Foundation zien. Zo is de bacterie- en schimmelgemeenschap in de bodem heel anders dan die in de mest. In de mest en melk was er een verschil te zien tussen gangbare en biologische bedrijven.

The number of bacteria that are resistant to antimicrobials is increasing worldwide. In the Netherlands, the number of resistant bacteria that can cause infections in humans has remained broadly stable. Nevertheless there is cause for concern and caution. Compared to 2015, in 2016 more 'outbreaks' in healthcare institutions of bacteria that are resistant to last-resort antimicrobials were reported. There is a chance that these bacteria will become more and more common. Although healthy people are not affected, these bacteria can make vulnerable people very sick. If more and more bacteria become resistant to antimicrobials, the treatment options will eventually become limited and it will also become more difficult to treat less serious conditions such as urinary tract infections. The more antimicrobials are used, the greater the chance that bacteria will develop resistance. In 2016, general practitioners wrote approximately two percent fewer prescriptions for antimicrobials than in 2015. The total use of antimicrobials in Dutch hospitals remained stable in 2015, compared to an increase in antimicrobial use in the previous year. The use of antimicrobials for animals decreased further in 2016 compared to 2015, but has been decreasing more slowly in recent years than it did previously. The degree of bacterial resistance in animals also decreased further. This is shown in the annual NethMap/MARAN 2017 report, in which various organisations present their data on antimicrobial use and resistance in the Netherlands, for humans as well as animals. Firstly, to combat resistance, it is important to base the choice to prescribe antimicrobials on the individual patient and the infection concerned. Secondly, it is important that it quickly becomes clear when resistant bacteria are involved and that proper tests are used to determine this. Thirdly, it is important that healthcare providers carefully follow existing hygiene procedures, such as handwashing, in order to prevent resistant bacteria from spreading. For example, thanks to these measures, the number of MRSA bacteria in hospitals has remained low in recent years. This type of 'hospital bacteria' is transmitted via skin-to-skin contact, particularly via the hands, and is insensitive to many types of antimicrobials. Part 1: NethMap 2017 pg 1 - 160 Part 2: MARAN 2017 pg 1 - 80

The number of bacteria that are resistant to antimicrobials is increasing worldwide. In the Netherlands, the number of resistant bacteria that can cause infections in humans has remained broadly stable. Nevertheless there is cause for concern and caution. Compared to 2015, in 2016 more 'outbreaks' in healthcare institutions of bacteria that are resistant to last-resort antimicrobials were reported. There is a chance that these bacteria will become more and more common. Although healthy people are not affected, these bacteria can make vulnerable people very sick. If more and more bacteria become resistant to antimicrobials, the treatment options will eventually become limited and it will also become more difficult to treat less serious conditions such as urinary tract infections. The more antimicrobials are used, the greater the chance that bacteria will develop resistance. In 2016, general practitioners wrote approximately two percent fewer prescriptions for antimicrobials than in 2015. The total use of antimicrobials in Dutch hospitals remained stable in 2015, compared to an increase in antimicrobial use in the previous year. The use of antimicrobials for animals decreased further in 2016 compared to 2015, but has been decreasing more slowly in recent years than it did previously. The degree of bacterial resistance in animals also decreased further. This is shown in the annual NethMap/MARAN 2017 report, in which various organisations present their data on antimicrobial use and resistance in the Netherlands, for humans as well as animals. Firstly, to combat resistance, it is important to base the choice to prescribe antimicrobials on the individual patient and the infection concerned. Secondly, it is important that it quickly becomes clear when resistant bacteria are involved and that proper tests are used to determine this. Thirdly, it is important that healthcare providers carefully follow existing hygiene procedures, such as handwashing, in order to prevent resistant bacteria from spreading. For example, thanks to these measures, the number of MRSA bacteria in hospitals has remained low in recent years. This type of 'hospital bacteria' is transmitted via skin-to-skin contact, particularly via the hands, and is insensitive to many types of antimicrobials. Part 1: NethMap 2017 pg 1 - 160 Part 2: MARAN 2017 pg 1 - 80

Wereldwijd neemt het aantal bacteriën die resistent zijn tegen antibiotica toe. In Nederland blijft dat aantal over het algemeen stabiel en is het minder hoog dan in veel andere landen. Toch blijft er reden voor zorg en alertheid. Bij sommige bacteriesoorten neemt de resistentie tegen sommige antibiotica wel langzaam toe. Vooral bij Klebsiella pneumoniae, een veel voorkomende darmbacterie, werken de laatste 5 jaar meerdere antibiotica steeds vaker minder goed. Deze bacteriën kunnen onschuldige infecties zoals een blaasontsteking veroorzaken en zijn door de resistentie moeilijker te behandelen. Ook moeten dan vaker soorten antibiotica worden gebruikt die alleen als laatste redmiddel worden gebruikt. Om resistentie te voorkomen is het belangrijk om antibiotica op de juiste manier te gebruiken en alleen als het nodig is. Huisartsen schreven in het afgelopen jaar even veel antibioticakuren voor als de jaren daarvoor. In ziekenhuizen blijft het totale antibioticagebruik wel stijgen. Voor dieren is in 2018 is ongeveer evenveel antibiotica voorgeschreven als in 2017. Ten opzichte van 2009, het referentiejaar, is het gebruik met ruim 63 procent verminderd. Voor dieren zijn de afgelopen jaren bijna geen antibiotica gebruikt die belangrijk zijn om infecties bij de mens te behandelen. Het aantal resistente bacteriën bij dieren is ongeveer gelijk gebleven. Wel is het aantal ESBL-producerende darmbacteriën verder afgenomen bij bijna alle diersoorten die voor de voedselproductie worden gebruikt. Alleen bij vleeskalveren blijft het aantal stijgen. ESBL zijn enzymen die veelgebruikte antibiotica kunnen afbreken, zoals penicillines. Dit blijkt uit de jaarlijkse rapportage NethMap/MARAN 2019. Hierin presenteren diverse organisaties gezamenlijk de gegevens over het antibioticagebruik en -resistentie in Nederland, zowel voor mensen als voor dieren. In Nederland zijn de afgelopen jaren extra maatregelen genomen om antibioticaresistentie te bestrijden. Deze maatregelen reiken verder dan de gezondheidszorg omdat resistente bacteriën ook bij dieren, in voeding en in het milieu voorkomen (One Health). Onder andere zijn 'regionale zorgnetwerken' opgezet om de samenwerking tussen verschillende zorgprofessionals te stimuleren en de kans dat resistente bacteriën worden overgedragen zo klein mogelijk te houden. Part 1: NethMap 2019 pg 1 - 166 Part 2: MARAN 2019 pg 1 - 82

Wereldwijd neemt het aantal bacteriën die resistent zijn tegen antibiotica toe. In Nederland blijft dat aantal over het algemeen stabiel en is het minder hoog dan in veel andere landen. Toch blijft er reden voor zorg en alertheid. Bij sommige bacteriesoorten neemt de resistentie tegen sommige antibiotica wel langzaam toe. Vooral bij Klebsiella pneumoniae, een veel voorkomende darmbacterie, werken de laatste 5 jaar meerdere antibiotica steeds vaker minder goed. Deze bacteriën kunnen onschuldige infecties zoals een blaasontsteking veroorzaken en zijn door de resistentie moeilijker te behandelen. Ook moeten dan vaker soorten antibiotica worden gebruikt die alleen als laatste redmiddel worden gebruikt. Om resistentie te voorkomen is het belangrijk om antibiotica op de juiste manier te gebruiken en alleen als het nodig is. Huisartsen schreven in het afgelopen jaar even veel antibioticakuren voor als de jaren daarvoor. In ziekenhuizen blijft het totale antibioticagebruik wel stijgen. Voor dieren is in 2018 is ongeveer evenveel antibiotica voorgeschreven als in 2017. Ten opzichte van 2009, het referentiejaar, is het gebruik met ruim 63 procent verminderd. Voor dieren zijn de afgelopen jaren bijna geen antibiotica gebruikt die belangrijk zijn om infecties bij de mens te behandelen. Het aantal resistente bacteriën bij dieren is ongeveer gelijk gebleven. Wel is het aantal ESBL-producerende darmbacteriën verder afgenomen bij bijna alle diersoorten die voor de voedselproductie worden gebruikt. Alleen bij vleeskalveren blijft het aantal stijgen. ESBL zijn enzymen die veelgebruikte antibiotica kunnen afbreken, zoals penicillines. Dit blijkt uit de jaarlijkse rapportage NethMap/MARAN 2019. Hierin presenteren diverse organisaties gezamenlijk de gegevens over het antibioticagebruik en -resistentie in Nederland, zowel voor mensen als voor dieren. In Nederland zijn de afgelopen jaren extra maatregelen genomen om antibioticaresistentie te bestrijden. Deze maatregelen reiken verder dan de gezondheidszorg omdat resistente bacteriën ook bij dieren, in voeding en in het milieu voorkomen (One Health). Onder andere zijn 'regionale zorgnetwerken' opgezet om de samenwerking tussen verschillende zorgprofessionals te stimuleren en de kans dat resistente bacteriën worden overgedragen zo klein mogelijk te houden. Part 1: NethMap 2019 pg 1 - 166 Part 2: MARAN 2019 pg 1 - 82

In the past years, most chemicals and energy were produced from fossil-based resources. The continuous dependency on these depleting fossil resources have negatively impacted the environment with the emission of greenhouse gases and harmful materials. The replacement of fossil-based technologies to sustainable production methods such as those using microbes and fermentation has become a key research area. Non‐model thermophilic clostridia with native ability to ferment lignocellulose biomass, make them potential production organisms for industrial applications. However, the limitation of genetic accessibility and genetic tools for clostridial manipulation pose a major barrier for exploiting them as platform organisms. This thesis explores the genetic accessibility and tool development for two thermophilic clostridia, the ethanol-producing Hungateiclostridium thermocellum and the succinate- producing Pseudoclostridium thermosuccinogenes. In addition, we studied aspects such as xylose consumption, sporulation and CRISPRi-mediated silencing of central metabolism to improve strain engineering.For industrial strain development high engineering efficiencies are desired. Due to the user-friendliness and stringency, CRISPR-Cas-based technologies have strongly enhanced strain engineering efforts in bacteria. This has enabled more rapid metabolic engineering of both the model and non-model organisms, opening new possibilities to use them as improved cell factories. The discovery of novel Cas9-like systems from diverse microbial environments will extend the repertoire of applications and broaden the range of hosts to create novel platform organisms to produce biotechnologically relevant products.H. thermocellum DSM1313 has biotechnological potential as a whole-cell biocatalyst for ethanol production using lignocellulosic renewable sources. The full exploitation of H. thermocellum has been limited with the lack of high-throughput genome engineering tools. A thermophilic bacterial CRISPR- Cas9‐based system, that has been recently developed in our research group, was applied in the organism as a transcriptional gene repression tool. The ThermoCas9-based CRISPR interference (CRISPRi) was utilized to modulate the central metabolic lactate dehydrogenase (ldh) and phosphotransacetylase (pta) genes in H. thermocellum. The effects of gene repression were studied based on transcriptional expression and product formation. Single-guide RNA (sgRNA) under the control of native intergenic 16S/23S rRNA promoter from H. thermocellum directing the ThermodCas9 to the promoter region of both pta and ldh silencing transformants reduced expression up to 67% and 62%, correspondingly. This resulted in 24% and 17% decrease in lactate and acetate production, respectively. Hence, these data established for the first time, employing CRISPRi-mediated gene repression of metabolic genes in H. thermocellum can be used for remodelling of metabolic pathways without the requisite for genetic engineering.P. thermosuccinogenes DSM5809 is a thermophilic bacterium capable of producing succinate from lignocellulosic-derived sugars and has the potential to be exploited as a production organism. However, exploitation of P. thermosuccinogenes has been constrained partly due to the genetic inaccessibility and lack of genetic tools. In this study, we established the genetic accessibility of the organism by overcoming restriction barriers with in vivo methylation of plasmid DNA when transformed into an engineered E. coli HST04 strain expressing four native methylation systems of the thermophile. Transformation efficiencies of 102 CFU/µg plasmid DNA were achieved. The protocol was used to introduce a ThermodCas9-based CRISPRitool targeting the gene encoding malic enzyme in P. thermosuccinogenes, which resulted in a 75% downregulation of its expression. Additionally, the silencing of malic enzyme had an impact on the strain’s fermentation profile. This is the first example of genetic engineering in P. thermosuccinogenes, opening new possibilities for metabolic engineering of this bacterium.Apart from the development of genetic tools, this thesis also deals with heterologous expression of genes from P. thermosuccinogenes into H. thermocellum. The latter has been of interest for consolidated bioprocessing (CBP), because of its capability to ferment cellulose without any pre-treatment or additional enzymes. H. thermocellum has excellent cellulolytic activity for C6 substrates, but has the inability to grow on C5 substrates such as xylose. A GTP-dependent plasmid-based xylose gene cluster from P. thermosuccinogenes was introduced into H. thermocellum. The expression of the xylose utilization genes allowed for growth on xylose. Transcriptional analysis showed upregulation of the xylulokinase (xylB) and transcriptional regulator (xylR) genes on xylose compared to cellobiose. Unpredictably, introduction of the plasmid-based xylose gene cluster into H. thermocellum also impacted the cellobiose fermentation profile. Ethanol and acetate production was improved by 24% and 19%, respectively, for the xylose-plasmid bearing cells compared to the empty vector. To conclude, introducing the P. thermosuccinogenes xylose gene cluster is a useful step towards CBP with H. thermocellum to produce fuels and high value chemicals.Single-cell analysis of microbial population heterogeneity is a fast-growing research area in industrial biotechnology, environmental biology and pathogenesis due to its potential to identify and quantify the impact of subpopulations on microbial performance. Though several tools have been established, determination of population heterogeneity in anaerobic bacteria, especially spore-forming clostridia species has been studied thoroughly. We applied single cell analysis techniques such as flow cytometry (FCM) and fluorescence-assisted cell sorting (FACS) on the spore-forming succinate producer P. thermosuccinogenes. By combining FCM and FACS with fluorescent staining, we discriminated and enriched all sporulation-related morphologies of the thermophile. To evaluate the presence of metabolically active vegetative cells, a blend of the dyes propidium iodide (PI) and carboxy fluorescein diacetate (cFDA) tested best. Side scatter (SSC-H) in combination with metabolic indicator cFDA dye provided the best separation of sporulation populations. Based on this protocol, we successfully determined sporulation dynamics of P. thermosuccinogenes by distinguishing between spores, forespores, dark and bright phase endospores, and vegetative cells populations. Henceforth, this methodology can be applied to further study population heterogeneity and its impact on fermentation performance in the clostridia.In conclusion, this PhD thesis focuses on the two non-model thermophilic organisms as bacterial cell factories. Taking the succinate producer P. thermosuccinogenes as a model, we described strategies to overcome restriction barriers by in vivo methylation of plasmid DNA to have a genetically accessible strain. As a proof-of-concept, CRISPRi silencing tool was developed for both the thermophilic clostridia and applied to impact their transcriptional expression and fermentation profiles. Lignocellulolytic capacitiesofH.thermocellumwere improvedbytheintroductionofxylosegenesfrom P. thermosuccinogenes.Besides, tools weredevelopedtostudysporulationdynamics of P. thermosuccinogenes for future optimization of industrial fermentations.

De samenstelling van de bacterie- en schimmelgemeenschap als geheel is specifiek voor elk onderdeel van de kringloop op een melkveebedrijf. Dat laat een verkennend onderzoek gefinancierd door de Triodos Foundation zien. Zo is de bacterie- en schimmelgemeenschap in de bodem heel anders dan die in de mest. In de mest en melk was er een verschil te zien tussen gangbare en biologische bedrijven.

Bijdrage aan Zpannend Zernike

Fossil fuels, including oil, natural gas, and coal are primary energy sources and account for 86% of the world’s energy consumption. These fossil fuels are also used as a feedstock for the production of chemicals and materials. It took millions of years to form these fuels from biomass and their consumption is therefore considered non-renewable. At a certain moment the reserves of these fossil resources will be depleted and prior to that moment mining for bulk use will be economically unfeasible. Beside the finite reserves there are also many negative effects on the environment. These include the pollution of the environment and global warming. It is generally considered that global warming is caused by the release of greenhouse gasses, such as CH4, NOx, water vapour and CO2 into the atmosphere. Alternative resources for the generation of heat and power can be formed by geothermal, water, wind, and solar energy. However, these resources do not form an alternative for the production of chemicals and materials since this requires (chemical) building blocks. Biomass can form a suitable alternative resource for the formation of these building blocks. Biomass in its pure form is complex and can in most instances not directly be used for the production of renewable compounds. Just like fossil fuels it should be processed or biorefined to obtain building blocks for chemicals and materials. In the biorefining process all the non-food fractions such as the oils, gums, carbohydrates, proteins, and lignin should be used for their most energetically favourable applications. Nitrogen containing molecules are used in many chemical applications like the production of nylon, polyurethane and acrylonitrile. Naphtha does not comprise nitrogen-containing molecules and they have to be synthesised by binding nitrogen derived from air, a very energy intensive process. Forty percent of the integral cost price of nitrogen containing chemicals from fossil resources is formed by the energy and process costs. Biomass, on the other hand, contains nitrogen containing chemicals in the form amino acids. Traditionally, commercially available amino acids are obtained through microbial fermentations. These production volumes are generally low and the price is too high for bulk use. Therefore, alternative sources need to be investigated which can supply large volumes of amino acids at low cost. Interesting sources are the residual protein streams from the food industry or the production of biofuels. These streams are however heterogeneous in composition and result in low amino acid concentrations. Cyanophycin (CPG) is naturally accumulated by cyanobacteria as insoluble granules which form an energy and nitrogen reserve material and consists of equimolar amounts of arginine and aspartic acid. The insoluble nature can be used to isolate and concentrate the granules. The wish to produce bioethanol from (lignocellulosic) waste streams coupled to the concentration and subsequent conversion of amino acids into CGP culminated into the N-ergy project. The research described in this thesis was focused on the production of cyanophycin (CGP) in the filamentous fungus Rhizopus oryzae. Chapter 2 is an in-depth analysis of the potential of R. oryzae for the production of platform chemicals and the genetic accessibility. Strains of R. oryzae are capable of producing a wide range of lignocellulosic hydrolysing enzymes and grow on a wide range of carbon sources. Using these carbon sources it is capable of producing L-(+)-lactic acid, ethanol with a fumaric acid. The yields using D-glucose are in excess of 85% of the theoretical yield for L-(+)-lactic acid and ethanol and over 65% for fumaric acid. The hydrolytic capacity and high yields of fermentation end products make this an interesting organism for the biobased economy. Though, genetic modification is challenging since the introduced DNA rarely integrates in the genome leading to mitotically unstable phenotype. In order to produce cyanophycin several cyanophycin synthetase encoding genes were introduced in the sequenced strain R. oryzae 99-880. The genes originated from Synechocystis sp. strain PCC6803 and Anabaena sp. strain PCC7120. Also, a codon optimized version of the latter gene was introduced (chapter 3). Only one transformant (expressing chpA6803) out of a total of 92 isolates was able to produce water-soluble CGP (0.5%) with traces of water-insoluble CGP. There was no correlation between transcript levels of cphA, enzyme activity, and CGP accumulation. In addition, the water soluble CGP did not contain L-lysine which is generally assumed to be the cause for the soluble behaviour. The total amount of CGP and enzyme activity is low in comparison the other genetically modified microorganisms expressing cphA’s. The common methods to determine the amino acid composition of protein samples require an acid hydrolysis followed by a derivatisation step with costly reactants. Cost effective derivatisation methods are available yet these require manual handling prior to the analysis of each individual sample. In order to analyse large volume of samples, a cost effective and fast new method was required. A new method was developed using acid hydrolysis and automated pro-column derivatisation using o-phthalaldehyde/ethanethiol reagent in combination with 9-fluorenylmethyl chloroformate (chapter 4). Due to the automated derivatisation in the injection needle the handling time is greatly reduced. Additionally, the run time for is sample using the UPLC method was 16 minutes which is a reduction of roughly 50% in comparison to a HPLC separation. The method was used for the analysis of proteins like CGP and has a mean recovery of the amino acids of 95%. With the current available techniques, it is not possible to further reduce the time required for the derivatisation and analysis. Introduction of CphA is R. oryzae was successful but resulted only in the accumulation of trace amounts of soluble cyanophycin. A possible explanation for the low accumulation is the fact that the production of soluble cyanophycin has a detrimental effect on growth of R. oryzae. We therefore tested the introduction of heterologous genes that should have a beneficial effect on growth. R. oryzae 99-880 has no xylanolytic capacity although it can grow on xylose. In order to increase the xylanolytic activity a β-1,4-endoxylanase encoding gene (xynB) of Aspergillus niger CBS 513.88 was introduced (chapter 5) using the same method as employed for the introduction of cphA’s. In total 13 transformants were isolated of which three displayed enzyme activity and one was mitotically stable. In this experiment there is a clear correlation between transcript levels of xynB and enzyme activity. The stable transformant was able to grow on xylan as carbon source. This demonstrated that metabolic engineering using the selected transformation system can be performed successfully. The large difference in numbers between transformants that successful produce β-1,4-endoxylanase in comparison to cyanophycin is indicative that the bottleneck in CGP production is product related. In recent years little development is published in respect to the genetic engineering of R. oryzae (chapter 6). The systems that are published are not always used to express genes and some are developments from the system that was also used in this study. Still R. oryzae is widely studied as an organism of interest for the biobased economy due to the hydrolytic capacity and range of product that can be formed by the various strains.

Wereldwijd neemt het aantal bacteriën die resistent zijn tegen antibiotica toe. In Nederland is dat aantal over het algemeen ongeveer stabiel gebleven. Toch blijft er reden voor zorg en oplettendheid. Zowel bij patiënten van huisartsen als van ziekenhuizen komen bepaalde resistente bacteriën de afgelopen 5 jaar vaker voor, de zogeheten ESBL-producerende darmbacteriën. ESBL zijn enzymen die veelgebruikte antibiotica kunnen afbreken, zoals penicillines. Deze bacteriën kunnen onschuldige infecties zoals een blaasontsteking veroorzaken die door de resistentie moeilijker te behandelen zijn. Ook moet dan vaker gebruik worden gemaakt van soorten antibiotica die alleen als laatste redmiddel worden ingezet. Om resistentie te voorkomen is het belangrijk om antibiotica op de juiste manier te gebruiken en alleen als het nodig is. In de afgelopen jaren schreven huisartsen minder antibioticakuren voor. In ziekenhuizen daarentegen steeg het totale antibioticagebruik in 2016 ten opzichte van het voorgaande jaar. Het totale antibioticagebruik voor dieren was in 2017 per saldo vergelijkbaar met 2016. In sommige diersectoren daalde het gebruik, terwijl het in andere sectoren licht toenam. Antibiotica die belangrijk zijn om infecties bij de mens te behandelen, zijn de afgelopen jaren nauwelijks meer voor dieren ingezet. Zo is het aantal ESBL's verder afgenomen bij bijna alle soorten dieren die voor de voedselproductie worden gebruikt. Een uitzondering daarop zijn vleeskalveren, waar een lichte toename is gezien. Dit blijkt uit de jaarlijkse rapportage NethMap/MARAN 2018. Hierin presenteren diverse organisaties gezamenlijk de gegevens over het antibioticagebruik en -resistentie in Nederland, zowel voor mensen als voor dieren. In de afgelopen twee jaar zijn in Nederland extra maatregelen genomen om antibioticaresistentie te bestrijden. Deze maatregelen reiken verder dan de gezondheidszorg. Resistente bacteriën houden zich immers niet aan landgrenzen en komen ook bij dieren, in voeding en in het milieu voor (One Health). Om deze aanpak te ondersteunen zijn in 2017 'regionale zorgnetwerken' opgezet. Zij hebben de taak om de samenwerking tussen verschillende zorgprofessionals te stimuleren bij het voorkomen en bestrijden van antibioticaresistentie. Daarnaast is er meer aandacht voor antibioticaresistentie in verpleeghuizen. Zo is een onderzoek gestart waarin wordt gemeten hoeveel bewoners resistente bacteriën bij zich dragen. De uitkomst hiervan wordt eind 2018 verwacht. Part 1: NethMap 2017 pg 1 - 156 Part 2: MARAN 2017 pg 1 - 78

Host-pathogen interactions are among the most prevalent and evolutionary important interactions known today. The predation of prokaryotes by their viruses is happening on an especially large scale and had a major influence on the evolutionary history of prokaryotes. Since most viruses are lytic at some point in their life-cycle, there is a high selection pressure for prokaryotes to develop defense mechanisms. As described in Chapter 1, the CRISPR-Cas system is a relatively recently discovered defense system and is also the first adaptive defense system discovered in prokaryotes. CRISPR-Cas systems are widespread, occurring in the majority of archaea and also a considerable fraction of bacteria. This diversity is also reflected in the diversity of different types of CRISPR-Cas systems, currently being divided into 6 major types with a large number of subtypes. The type I-E system of Escherichia coli is a well-studied model system and of high relevance, since it is a major subtype of type I systems which make up around 50 % of all discovered CRISPR-Cas systems. CRISPR-Cas systems basically comprise the CRISPR array, made up of repeats and foreign derived spacers, and a set of cas genes. Immunity is commonly divided into three functional stages, adaptation, expression and interference. Adaptation is the acquisition of new spacers from the foreign nucleic acid and its incorporation into the CRISPR array. During expression, the CRISPR array is transcribed, processed and assembled with Cas proteins into CRISPR RNA (crRNA) guided ribonucleoprotein complexes (crRNP). Interference is the detection, binding and destruction of foreign nucleic acids by the crRNP and in type I systems the Cas3 nuclease. The type I-E system contains another function, called primed adaptation. Primed adaptation is a more rapid and efficient version of regular (naïve) adaptation. In addition to the adaptation machinery, primed adaptation also requires the interference machinery. Chapter 2 describes and compares a fundamental feature of most, if not all, CRISPR-Cas systems and also many other small RNA based systems. The mode of action of small RNAs relies on protein-assisted base pairing of the guide RNA with target mRNA or DNA to interfere with their transcription, translation or replication. Several unrelated classes of small non-coding RNAs have been identified including eukaryotic RNA silencing associated small RNAs, prokaryotic small regulatory RNAs and prokaryotic CRISPR (clustered regularly interspaced short palindromic repeats) RNAs. All three groups identify their target sequence by base pairing after finding it in a pool of millions of other nucleotide sequences in the cell. In this complicated target search process, a region of 6 to 12 nucleotides of the small RNA termed the ‘seed’ plays a critical role. The seed is often a structurally pre-ordered region that increases accessibility and lowers the energy barrier of RNA-DNA duplex formation. Furthermore, the length of the seed is optimally chosen to allow rapid probing and also rejection of potential target sites. The seed is a perfect example of parallel evolution, showing that nature comes up with the same strategy independently multiple times. Chapter 3 provides a description and protocol of the Electrophoretic Mobility Shift Assay (EMSA) and its use for studying crRNPs. EMSA is a straightforward and inexpensive method for the determination and quantification of protein–nucleic acid interactions. It relies on the different mobility of free and protein-bound nucleic acid in a gel matrix during electrophoresis. Nucleic acid affinities of crRNPs can be quantified by calculating the dissociation constant (Kd ). Protocols for two types of EMSA assays are described using the Cascade ribonucleoprotein complex from Escherichia coli as an example. One protocol uses plasmid DNA as substrate, while the other uses short linear oligonucleotides. Plasmids can be easily visualized with traditional DNA staining, while oligos have to be radioactively labelled using the 32Phosphate isotope. The EMSA method and these protocols are applied throughout the other chapters of this thesis. Chapter 4 focusses on the processes of interference and primed adaptation, specifically on their tolerance of mutations. Invaders can escape Type I-E CRISPR-Cas immunity in E. coli by making point mutations in the protospacer (especially in the seed) or its adjacent motif (PAM), but hosts quickly restore immunity by integrating new spacers in a positive feedback process termed priming. Here, we provide a systematic analysis of the constraints of both direct interference and subsequent priming in E. coli. We have defined a high-resolution genetic map of direct interference by Cascade and Cas3, which includes five positions of the protospacer at 6 nt intervals that readily tolerate mutations. Importantly, we show that priming is an extremely robust process capable of utilizing degenerate target regions with up to at least eleven mutations throughout the PAM and protospacer region. Priming is influenced by the number of mismatches, their position and is nucleotide dependent. Our findings imply that even out-dated spacers containing many mismatches can induce a rapid primed CRISPR response against diversified or related invaders, giving microbes an advantage in the co- evolutionary arms race with their invaders. In Chapter 5 we elucidate the mechanism of priming. Specifically, we determine how new spacers are produced and selected for integration into the CRISPR array during priming. We show that priming is directly dependent on interference. Rapid priming occurs when the rate of interference is high, delayed priming occurs when the rate of interference is low. Using in vitro assays and next generation sequencing, we show that Cas3 couples CRISPR interference to adaptation by producing DNA breakdown products that fuel the spacer integration process in a two-step, PAM-associated manner. The helicase-nuclease Cas3 pre-processes target DNA into fragments of about 30–100 nt enriched for thymine-stretches in their 3’ ends. By reconstituting the spacer integration process in vitro, we show that the Cas1-2 complex further processes these fragments and integrates them sequence- specifically into CRISPR repeats by coupling of a 3’ cytosine of the fragment. Our results highlight that the selection of PAM-compliant spacers during priming is enhanced by the combined sequence specificities of Cas3 and the Cas1-2 complex, leading to an increased propensity of integrating functional CTT-containing spacers. In Chapter 6 we look deeper into a nucleotide specific effect on priming that was discovered in Chapter 4. Immunity is based on the complementarity of host encoded spacer sequences with protospacers on the foreign genetic element. The efficiency of both direct interference and primed acquisition depends on the degree of complementarity between spacer and protospacer. Previous studies focused on the amount and positions of mutations, not the identity of the substituted nucleotide. In Chapter 4, we describe a nucleotide bias, showing a positive effect on priming of C substitutions and a negative effect on priming of G substitutions in the basepairing strand of the protospacer. Here we show that these substitutions rather directly influence the efficiency of interference and therefore indirectly influence the efficiency of interference dependent priming. We show that G substitutions have a profoundly negative effect on interference, while C substitutions are readily tolerated when in the same positions. Furthermore, we show that this effect is based on strongly decreased binding of the effector complex Cascade to G mutants, while C mutants only minimally affect binding. In Chapter 5 we showed a connection between the rate of interference and the time of occurrence of priming. Here, we also quantify the extent of priming and show that priming is very prevalent in a population that shows intermediate levels of interference, while high or low levels of interference lead to a lower prevalence of priming. Chapter 7 describes an attempt to make use of our knowledge about the Cascade complex and develop it into a genome editing tool. The development of genome editing tools has made major leaps in the last decade. Recently, RNA guided endonucleases (RGENs) such as Cas9 or Cpf1 have revolutionized genome editing. These RGENs are the hallmark proteins of class II CRISPR-Cas systems. Here, we have explored the possibility to develop a new genome editing tool that makes use of the Cascade complex from E. coli. This RNA guided protein complex is fused to a FokI nuclease domain to sequence specifically cleave DNA. We validate the tool in vitro using purified protein and two sets of guide RNAs, showing specific cleavage activity. The tool requires two target sites of 32 nt each at a distance of 30-40 nt and inward facing three nucleotide flexible PAM sequences. Cleavage occurs in the middle between the two binding sites and primarily creates 4 nt overhangs. Furthermore, we show that an additional RFP can be fused to FokI-Cascade, allowing visualization of the complex in target cells. Unfortunately, we were not able to successfully apply the tool in vivo in eukaryotic cells.

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.