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

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

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

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

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

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

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

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

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

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

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

Sulphate can be removed from wastewater by means of biological anaerobic reduction to sulphide. The reduction requires the presence of a substrate that can serve as an electron donor. Methanol is a suitable electron donor for sulphate reduction under thermophilic conditions. In an anaerobic system containing methanol and sulphate, acetogenic bacteria (AB) and methanogenic archaea (MA) compete with sulphate reducing bacteria (SRB) for methanol or its degradation intermediates. Previously obtained results indicate that at 65°C SRB and MA mainly compete for the intermediate hydrogen instead of methanol. For efficient use of methanol as electron donor for sulphate reduction it is important that for the treatment of sulphate wastewater in an anaerobic reactor SRB out-compete MA. The mechanisms that determine the outcome of the competition are, however, not well understood. This paper describes a model based on growth kinetics of methanol-oxidising AB, and hydrogen-consuming SRB and MA, that can describe the competition between SRB and MA in a methanol-fed bioreactor. We present the model and its calibration using experimental data, and we discuss its shortcomings and suggest possible improvements.

The syntrophic propionate-oxidizing bacterium Syntrophobacter fumaroxidans possesses two distinct formate dehydrogenases and at least three distinct hydrogenases. All of these reductases are either loosely membrane-associated or soluble proteins and at least one of the hydrogenases is located in the periplasm. These enzymes were expressed on all growth substrates tested, though the levels of each enzyme showed large variations. These findings suggest that both H2 and formate are involved in the central metabolism of the organism, and that both these compounds may serve as interspecies electron carriers during syntrophic growth on propionate.

Salmonella is bij iedereen bekend. Zo niet campylobacter. Ten onrechte: de bacterie zorgt voor gezondheidsproblemen bij de mens. Soms in hevige mate. Onderzoek naar verbetering moet komen. Dit artikel geeft de stand van zaken weer

Bij de twee Nederlandse destructiebedrijven werd in 1993 onderzoek uitgevoerd naar de effectiviteit van de autoclaveringsprocessen en de microbiologische gesteldheid van bedrijfsruimten en eindprodukten. Bij de bedrijven N en C werden sporen van sulfiet reducerende clostridia aangetoond in respectievelijk 3,1% en 9,2% van de monsters destructiemateriaal, genomen direct na de autoclavering. In een monster, van bedrijf N, betrof het een besmetting met sporen van C.perfringens. De aanwezigheid van sporen in het geautoclaveerde destructiemateriaal duidt erop dat het autoclaveringsproces niet altijd leidt tot steriliteit. In monsters eindprodukt werd geen Salmonella aangetoond. In alle eindproduktmonsters was het Enterobacteriaceae-kiemgetal kleiner dan 10 kiemen per gram. Bij de bedrijven N en C werd Salmonella aangetoond in respectievelijk 36,7% en 13,3% van monstsers stof uit bedrijfsruimten. In 23,3% van de stofveegmonsters uit bedrijf N was het Enterobacteriaceae-kiemgetal groter dan 300 kiemen per gram.

Studies were carried out in 1991 on the efficacy of sterilisation processes at three rendering plants for dead animals and animal wastes in the Netherlands. Spores of sulfite reducing clostridia were detected in 4.8% of the samples taken directly after autoclaving. However, C.perfringens was not detected. Salmonella was detected in one sample (5%) of final products from plant C, and no Salmonella was detected in samples of final products from plants A and B. At plants A and C Salmonella was detected in 7.4% and 65.0% respectively of samples of dust taken from the processing halls. At plant B no dust samples were examined. In 93.5% of samples of final products the number of Enterobacteriaceae was less than 10 cfu per gram. Higher numbers of Enterobacteriaceae were detected incidentally in samples of final products from plants A and B and in dust samples from plant A, but frequently in dust samples from plant C. The results indicate poor hygienic conditions at plant C. This plant has been closed down in june 1991.

Studies were carried out in 1990 on the efficacy of sterilisation processes at three rendering plants for dead animals and animal wastes in the Netherlands. Spores of sulfite reducing clostridia were detected in 11.8% of the samples taken directly after autoclaving. C.perfringens was detected in 1.2% of the samples from plant A and in 2.9% of the samples from plant C. At plants A, B and C Salmonella was detected in 0%, 2.2% and 20.0% respectively of samples of final products and in 12.0%, 9.1% and 80.0% respectively of samples of dust taken from the processing halls. In all but one samples of final products the number of Enterobacteriaceae was less than 10 cfu per gram. Higher numbers of Enterobacteriaceae were detected incidentally in dust samples from plants A and B, but frequently in dust samples from plant C. This result suggests that the contamination of final products at plant C was due to poor hygienic conditions.

Studies carried out in 1989 on the efficacy of rendering processes in the Netherlands in the elimination of microbiological agents, showed that spores of Clostridium were present in 12,2% of the samples taken directly after autoclaving. C.perfringens was detected in less than 2% of the samples. In samples of final products no Salmonella was detected and the number of Enterobacteriaceae was

At two rendering plants for dead animals and animal wastes in the Netherlands studies were carried out in 1992 on the efficacy of autoclaving processes and the microbiological condition of processing halls and final products. At plants A and B spores of sulfite reducing clostridia were detected in 3,3% and 11,5% respectively of the samples taken directly after autoclaving. In one sample, from plant A, spores of C.perfringens were concerned. In samples of final product Salmonella was not detected. In all but one of these samples the Enterobacteriaceae-count was less than 10 cfu per gram. At plants A and B Salmonella was detected in 36,7% and 3,7% respectively of samples of dust taken from the processing halls. In 30,0% of samples of dust from plant A the Enterobacteriaceae-count exceeded 300 cfu per gram.

rapport met CD-rom

The microbial reduction of chlorate and perchlorate has been known for long as a respiratory process of mesophilic bacteria that thrive in diverse environments such as soils, marine and freshwater sediments. Chlorate and perchlorate are found in nature deriving from anthropogenic and natural sources and can, in the absence of oxygen, be reduced by respective microorganisms to chloride coupled to energy conservation and growth. These classical chlorate- and perchlorate-reducing microorganisms employ enzymes that reduce perchlorate (or chlorate) to the intermediate chlorite, followed by the disproportionation of chlorite to chloride and dioxygen. The latter has been regarded as key reaction for complete (per)chlorate reduction, catalyzed by the enzyme chlorite dismutase, which forms oxygen under anaerobic conditions. This de novo produced oxygen is reduced by terminal oxidases in the metabolism of facultative anaerobic (per)chlorate-reducing microorganisms and can be used by oxygenases for the activation of recalcitrant substrates, as was shown earlier for hydrocarbons. The potentially stimulating effect of chlorate and perchlorate on microorganisms indigenous to petroleum reservoirs was discussed, seeking new strategies for microbial enhanced oil recovery (based on subsurface growth stimulation and partial hydrocarbon degradation) and reservoir souring control (by inhibiting sulfate-reducing prokaryotes and diminishing sulfide formation). This thesis reports the capability of hyperthermophilic and thermophilic prokaryotes that originate from subsurface environments to grow by the reduction of chlorate and/or perchlorate. In contrast to the classical metabolism of mesophilic (per)chlorate-reducing microorganisms this study demonstrated that a chlorite-disproportionating enzyme is commonly absent in (hyper)thermophilic (per)chlorate reducers. The absence of this enzyme that was previously defined as prerequisite for (per)chlorate reduction is overcome by the chemical reactivity of reduced sulfur compounds with chlorite generated. In the here more closely investigated hyperthermophilic archaea (Archaeoglobus fulgidus and Aeropyrum pernix) and thermophilic Firmicutes (Carboxydothermus hydrogenoformans and Moorella glycerini strain NMP) chlorite is formed by the activity of molybdopterin oxidoreductases. The respective enzymes are remotely related to perchlorate reductases of mesophilic bacteria and nitrate reductases of the bacterial Nar-type. In contrast to classical bacterial Nar-type enzymes, above-mentioned enzymes seem to have their catalytic subunits outside of the cell. As a consequence the reduction of (per)chlorate forms chlorite extracellularly where it reacts with reduced sulfur species present in the medium/environment (e.g. sulfide), forming chloride anions and oxidized sulfur species (SxOyz-). The hyperthermophilic archaeon Archaeoglobus fulgidus reduces these chemically formed sulfur species concomitantly to (per)chlorate reduction, which regenerates sulfide for the continuous reduction of (per)chlorate. This interaction of biotic and abiotic reactions during (per)chlorate reduction establishes an intraspecies “sulfur loop” that enables complete reduction of perchlorate to chloride. Whereas A. pernix also relies on the chemical reactivity of chlorite with thiosulfate, this archaeon does not employ systems for regenerating the reducing agents biologically; which is reflected by the accumulation of sulfate during perchlorate reduction. The Crenarchaeon A. pernix, formerly known as a strictly aerobic microorganism, expands the trait of microbial (per)chlorate reduction up to 100°C. In addition to the intraspecies “sulfur loop” of A. fulgidus, there were indications that the reduction of perchlorate may also proceed syntrophically, as indicated by a thermophilic bacterial consortium. In the respective culture, it seems that one microorganism reduces perchlorate, forming chlorite, which is chemically reduced by sulfide to chloride anions and oxidized sulfur compounds. Another group of microorganisms uses the respective sulfur compounds as electron acceptors and regenerates sulfide. Sulfur (of different redox states) depicts the mediating agent in this interspecies “sulfur loop”, but may possibly be substituted in nature by other compounds such as ferrous/ferric iron. Here presented (per)chlorate reduction sensu lato, which lacks the action of a chlorite-disproportionating enzyme may be widely spread among prokaryotes. For example enzymes closely resembling the designated (per)chlorate-reducing enzyme in Archaeoglobus fulgidus are also found in other strictly anaerobic thermophiles, of which C. hydrogenoformans and M. glycerini NMP were already confirmed to grow by the reduction of (per)chlorate as well. The substrate ambiguity of particular periplasmic DMSO enzymes may enable a broader group of microorganisms of (per)chlorate reduction sensu lato, in case sulfide is present in the environment. A broadened substrate spectrum of respective enzymes (beyond their canonical function) may possibly have had evolutionary advantages. Chlorine oxyanions are naturally formed and have been introduced on Earth for ages already. The reduction of (per)chlorate and formation of chlorite in ancient anaerobic microorganisms may even have contributed to the evolution of proteins adapted to oxidizing conditions on early Earth and preceded the evolution of oxygenic photosynthesis. It is shown that subsurface-inhabiting (hyper)thermophiles are able to grow by the reduction of (per)chlorate, which is also of interest for applications in the field of oil recovery. The finding that (per)chlorate reduction is interfering with the sulfur metabolism of a major contributor to reservoir souring in hot oil fields, A. fulgidus, draws promising scenarios for future attempts in developing novel souring control strategies. (Per)chlorate reduction by A. fulgidus was also coupled to the oxidation of butyrate, a volatile fatty acid commonly present in petroleum reservoirs. For sustainable applications in the oil recovery business, it is desirable to rely, as little as possible, on external substrates. In this respect the fact that A. fulgidus couples (per)chlorate reduction to the oxidation of butyrate is advantageous. Possibly the microorganism can also degrade long-chain alkanes and alkenes coupled to (per)chlorate reduction, a feature that was shown earlier coupled to sulfate reduction. All together a shift of A. fulgidus from sulfate reduction to (per)chlorate reduction in oil fields would not only diminish souring, but maintain/stimulate in-situ growth of the microorganism (based on intrinsic carbon sources) which has additionally advantageous effects for improved sweeping efficiencies during water flooding.

The microbial reduction of chlorate and perchlorate has been known for long as a respiratory process of mesophilic bacteria that thrive in diverse environments such as soils, marine and freshwater sediments. Chlorate and perchlorate are found in nature deriving from anthropogenic and natural sources and can, in the absence of oxygen, be reduced by respective microorganisms to chloride coupled to energy conservation and growth. These classical chlorate- and perchlorate-reducing microorganisms employ enzymes that reduce perchlorate (or chlorate) to the intermediate chlorite, followed by the disproportionation of chlorite to chloride and dioxygen. The latter has been regarded as key reaction for complete (per)chlorate reduction, catalyzed by the enzyme chlorite dismutase, which forms oxygen under anaerobic conditions. This de novo produced oxygen is reduced by terminal oxidases in the metabolism of facultative anaerobic (per)chlorate-reducing microorganisms and can be used by oxygenases for the activation of recalcitrant substrates, as was shown earlier for hydrocarbons. The potentially stimulating effect of chlorate and perchlorate on microorganisms indigenous to petroleum reservoirs was discussed, seeking new strategies for microbial enhanced oil recovery (based on subsurface growth stimulation and partial hydrocarbon degradation) and reservoir souring control (by inhibiting sulfate-reducing prokaryotes and diminishing sulfide formation). This thesis reports the capability of hyperthermophilic and thermophilic prokaryotes that originate from subsurface environments to grow by the reduction of chlorate and/or perchlorate. In contrast to the classical metabolism of mesophilic (per)chlorate-reducing microorganisms this study demonstrated that a chlorite-disproportionating enzyme is commonly absent in (hyper)thermophilic (per)chlorate reducers. The absence of this enzyme that was previously defined as prerequisite for (per)chlorate reduction is overcome by the chemical reactivity of reduced sulfur compounds with chlorite generated. In the here more closely investigated hyperthermophilic archaea (Archaeoglobus fulgidus and Aeropyrum pernix) and thermophilic Firmicutes (Carboxydothermus hydrogenoformans and Moorella glycerini strain NMP) chlorite is formed by the activity of molybdopterin oxidoreductases. The respective enzymes are remotely related to perchlorate reductases of mesophilic bacteria and nitrate reductases of the bacterial Nar-type. In contrast to classical bacterial Nar-type enzymes, above-mentioned enzymes seem to have their catalytic subunits outside of the cell. As a consequence the reduction of (per)chlorate forms chlorite extracellularly where it reacts with reduced sulfur species present in the medium/environment (e.g. sulfide), forming chloride anions and oxidized sulfur species (SxOyz-). The hyperthermophilic archaeon Archaeoglobus fulgidus reduces these chemically formed sulfur species concomitantly to (per)chlorate reduction, which regenerates sulfide for the continuous reduction of (per)chlorate. This interaction of biotic and abiotic reactions during (per)chlorate reduction establishes an intraspecies “sulfur loop” that enables complete reduction of perchlorate to chloride. Whereas A. pernix also relies on the chemical reactivity of chlorite with thiosulfate, this archaeon does not employ systems for regenerating the reducing agents biologically; which is reflected by the accumulation of sulfate during perchlorate reduction. The Crenarchaeon A. pernix, formerly known as a strictly aerobic microorganism, expands the trait of microbial (per)chlorate reduction up to 100°C. In addition to the intraspecies “sulfur loop” of A. fulgidus, there were indications that the reduction of perchlorate may also proceed syntrophically, as indicated by a thermophilic bacterial consortium. In the respective culture, it seems that one microorganism reduces perchlorate, forming chlorite, which is chemically reduced by sulfide to chloride anions and oxidized sulfur compounds. Another group of microorganisms uses the respective sulfur compounds as electron acceptors and regenerates sulfide. Sulfur (of different redox states) depicts the mediating agent in this interspecies “sulfur loop”, but may possibly be substituted in nature by other compounds such as ferrous/ferric iron. Here presented (per)chlorate reduction sensu lato, which lacks the action of a chlorite-disproportionating enzyme may be widely spread among prokaryotes. For example enzymes closely resembling the designated (per)chlorate-reducing enzyme in Archaeoglobus fulgidus are also found in other strictly anaerobic thermophiles, of which C. hydrogenoformans and M. glycerini NMP were already confirmed to grow by the reduction of (per)chlorate as well. The substrate ambiguity of particular periplasmic DMSO enzymes may enable a broader group of microorganisms of (per)chlorate reduction sensu lato, in case sulfide is present in the environment. A broadened substrate spectrum of respective enzymes (beyond their canonical function) may possibly have had evolutionary advantages. Chlorine oxyanions are naturally formed and have been introduced on Earth for ages already. The reduction of (per)chlorate and formation of chlorite in ancient anaerobic microorganisms may even have contributed to the evolution of proteins adapted to oxidizing conditions on early Earth and preceded the evolution of oxygenic photosynthesis. It is shown that subsurface-inhabiting (hyper)thermophiles are able to grow by the reduction of (per)chlorate, which is also of interest for applications in the field of oil recovery. The finding that (per)chlorate reduction is interfering with the sulfur metabolism of a major contributor to reservoir souring in hot oil fields, A. fulgidus, draws promising scenarios for future attempts in developing novel souring control strategies. (Per)chlorate reduction by A. fulgidus was also coupled to the oxidation of butyrate, a volatile fatty acid commonly present in petroleum reservoirs. For sustainable applications in the oil recovery business, it is desirable to rely, as little as possible, on external substrates. In this respect the fact that A. fulgidus couples (per)chlorate reduction to the oxidation of butyrate is advantageous. Possibly the microorganism can also degrade long-chain alkanes and alkenes coupled to (per)chlorate reduction, a feature that was shown earlier coupled to sulfate reduction. All together a shift of A. fulgidus from sulfate reduction to (per)chlorate reduction in oil fields would not only diminish souring, but maintain/stimulate in-situ growth of the microorganism (based on intrinsic carbon sources) which has additionally advantageous effects for improved sweeping efficiencies during water flooding.

Net zoals een inseminator, een voorlichter, een dierenarts of willekeurig welke bezoeker is de loonwerker een mogelijke verspreider van smetstof. Hoe zit dat dan met smetstof en mest? Mest als een bron van mogelijke versleep van besmetting tussen bedrijven. Wat kun je er aan doen om die risico's te elimineren of op zijn minst te beperken? Dit artikel geeft u een aantal oplossingen

Onderzoek is verricht naar de mogelijke toekomstige gezondheidsrisico's verbonden aan een toename in het gebruik van oppervlaktewater als bron voor drinkwater. Voor een aantal bestrijdingsmiddelen, zware metalen, micro-organismen en desinfectiebijproducten zijn de toekomstige gezondheidsrisico's bepaald met behulp van de ketenbenadering van emissie van deze stoffen en micro-organismen in het milieu tot en met de concentratie in drinkwater. Om inzicht te krijgen in het effect van de drinkwaterbereiding op de (toekomstige) drinkwaterkwaliteit zijn diverse zuiveringsmethoden doorgerekend. Geconcludeerd kan worden dat de kwaliteit van het oppervlaktewater met als functie drinkwater voor de beschouwde bestrijdingsmiddelen in zeer lichte mate zal verbeteren tot 2015, daarentegen zal er voor de zware metalen en micro-oganismen nauwelijks een verandering optreden tot 2020. Stoffen (bestrijdingsmiddelen en zware metalen) vormen nauwelijks een bedreiging voor de drinkwaterkwaliteit. Verwijdering van micro-organismen vereist een uitgebreide zuivering, waarbij een zuivering gebaseerd op bodempassage met gesloten terugwinning de meest gewenste methode is. Als alternatief kunnen technieken als membraanfiltratie en chemische desinfectie worden toegepast. De hoeveelheid gevormde desinfectiebijproducten overschrijdt in veel gevallen de desbetreffende risicogrenzen. Dit rapport geeft een momentopname van de kwaliteitsaspecten van een aantal zuiveringstypen volgens het 'worst-case' scenario. Het geeft aan welke factoren (stoffen/micro-organismen en zuiveringsstappen), gelet op de ontwikkeling van de ruwwaterkwaliteit, in de toekomst een rol zouden kunnen gaan spelen.

In het kader van de realisatie van de Nationale Milieuverkenning 2000-2030 is het infectierisico bepaald voor een aantal pathogene micro-organismen (enterovirussen, Cryptosporidium en Giardia) bij het gebruik van rijks- en regionale oppervlaktewateren als bron voor de bereiding van drinkwater. Dit rapport beschrijft de wijze waarop deze analyse is uitgevoerd. In Nederland wordt van de totale hoeveelheid drinkwater ongeveer eenderde deel bereid uit oppervlaktewater, meestal rijkswater. In de toekomst zal dit aandeel toenemen omdat de hoeveelheid grondwater bestemd voor drinkwaterproductie wordt gestabiliseerd en het watergebruik volgens de prognoses verder zal stijgen. Het overheidsbeleid is gericht op een kwaliteit oppervlaktewater waaruit met eenvoudige middelen drinkwater geproduceerd kan worden. Op basis van het huidige beleid zal de kwaliteit van het oppervlaktewater nauwelijks veranderen. In het algemeen is de grootste risicofactor voor de kwaliteit van drinkwater bereid uit oppervlaktewater het voorkomen van pathogene micro-organismen (o.a. virussen en protozoa). In dit onderzoek is het risico op infectie met enterovirussen, Cryptosporidium en Giardia bij toepassing van 19 huidige en toekomstige innamepunten en zuiveringslocaties voor de drinkwaterproductie bepaald. Bij gebruik van de rijkswateren blijkt dat een eenvoudige zuivering gebaseerd op alleen chemische desinfectie niet voldoet. Indien men uitgaat van een acceptabel infectierisico voor pathogenen van 1 geinfecteerde persoon per 10.000 inwoners per jaar, wordt deze eis overschreden. Een conventionele zuivering (met spaarbekkens) voldoet voor alle locaties aan de risico-eis voor enterovirussen, maar niet voor Cryptosporidium en Giardia op alle beschouwde locaties. De zuiveringstechnieken bodempassage en dubbele membraanfiltratie zijn voldoende om een veilige drinkwaterproductie te garanderen op alle onderzochte locaties. De concentraties van de onderzochte pathogenen zijn lager in de beschouwde regionale oppervlaktewateren dan in de rijkswateren. Een conventionele zuivering kan hier voldoende zijn voor veilig drinkwater. In de toekomst is dit mogelijk 1 van de factoren die regionale wateren als drinkwaterbron voor relatief kleine productie-eenheden aantrekkelijk maakt.

72 water samples from 22 different recreational sites in the Netherlands were investigated in 1989 and 1990 for enteric viruses, F-specific RNA bacteriophages, Salmonella, Campylobacter and conventional faecal indicator bacteria (thermotolerant coliforms and faecal streptococci). Results were compared with Dutch and EC guidelines for bathing water quality and possible relationships between different groups of microorganisms were analyzed. Enteroviruses were detected in numbers > 1 PFU/10 1 (EC guideline value) in 7/72 samples (9.7%), all influenced by discharges of secondary efffluent. In one of these samples, the number of enteroviruses was > 1 PFU/1 (Dutch guideline value). The numbers of enteroviruses were correlated with thermotolerant coliforms and FRNA-phages. Occurrence of Salmonella and Campylobacter did not correlate with any of the other microorganisms.

In dit rapport worden de resultaten weergegeven van het onderzoek verricht naar de mogelijkheden van de (toekomstige) inzet van regionale oppervlaktewateren voor de drinkwatervoorziening. Tevens is onderzoek verricht naar huidige en mogelijk toekomstige gezondheidsrisico's verbonden aan het huidige gebruik van regionaal oppervlaktewater voor de drinkwaterbereiding. Voor het onderzoek zijn negen regionale watersystemen geselecteerd en onderzocht op een aantal relevante aspecten: kwaliteit, kwantiteit, risico's en effecten. Met behulp van kaarten zijn deze aspecten voor elk onderzocht watersysteem zichtbaar gemaakt. Door inventarisatie van de aspecten is getracht de geschiktheid van de wateren aan te geven als mogelijke bron voor de drinkwaterproductie. Met behulp van de ketenbenadering van emissie van stoffen en micro-organismen in het milieu tot en met de concentratie in drinkwater zijn voor een aantal bestrijdingsmiddelen, zware metalen en micro-organismen de huidige en mogelijk toekomstige gezondheidsrisico's bepaald. Om inzicht te kunnen verwerven in het effect van de drinkwaterbereiding op de (toekomstige) drinkwaterkwaliteit zijn twee bestaande zuiveringen en de 'eenvoudige zuivering' doorgerekend. Gezondheidsrisico's zijn bepaald door de concentraties in drinkwater te vergelijken met drinkwaternormen en gezondheidskundige risicogrenzen. Geconcludeerd kan worden dat bestrijdingsmiddelen en zware metalen nauwelijks een bedreiging vormen voor de drinkwaterkwaliteit. Voor de micro-organismen is 'eenvoudige zuivering' onvoldoende om te kunnen garanderen dat het drinkwater voldoet aan de gezondheidskundige risicogrenzen. Bodempassage en ozonisatie zijn effectieve verwijderingsprocessen om micro-biologisch 'veilig' drinkwater te kunnen garanderen. Het aspect kwantiteit speelt een belangrijke rol bij waterwinning, waardoor slechts enkele van de onderzochte wateren werkelijk geschikt worden bevonden als bron voor de productie van drinkwater. Bij enkele van deze watersystemen is het mogelijk om drinkwaterwinning in combinatie met natuur te ontwikkelen. Het gebruik van huishoudwater en industriewater kan uitkomst bieden bij kwantitatief kleinschalige projecten.

Het model TAPWAT (Tool for the Analysis of the Production of drinking WATer), is ontwikkeld om de drinkwaterkwaliteit te beschrijven voor integrale studies in het kader van het planbureau Milieu en Natuur van het RIVM. Het model bestaat uit modules die de individuele zuiveringsstappen van het drinkwaterzuiveringsproces vertegenwoordigen. De zuiveringsstappen in TAPWAT worden voornamelijk gebruikt in systemen voor de behandeling van oppervlaktewater tot drinkwater. De huidige TAPWAT versie zoals in dit rapport beschreven bestaat uit modules gebaseerd op verwijderingspercentages en modules gebaseerd op proces of semi-empirische modelering. Het model moet in staat zijn om het gehele pad te beschrijven van bron tot en met infectierisico voor de bevolking (tenminste voor pathogenen). Aanbevolen wordt een plan van aanpak te maken waarin de bestaande en de nog missende modules, stoffen en micro-organismen geprioriteerd worden in de tijd evenals de noodzakelijke validatie van het model. De verbetering van de huidige versie van TAPWAT en de voorgestelde slag om het model geschikt te maken voor risicoanalyse ten behoeve van de volksgezondheid zal volgens het plan van aanpak uitgevoerd dienen te worden.

De waterkwaliteitseisen voor gietwater in de opkweek zijn hoog. De planten vormen immers de basis voor de vervolgteelten. Opkweekbedrijven moeten bovendien aan de emissiedoelstellingen van de kaderrichtlijn water in 2027 voldoen: een nulemissie van nitraat en fosfaat. Daarom is een goede zuiveringstechniek voor zowel micro-organismen als gewasbeschermingsmiddelen nodig.

Als een endoscoop niet goed schoongemaakt wordt bestaat de kans dat infecties van patiënt op patiënt worden overgedragen. UMCG-onderzoeker Julia Kovaleva bestudeerde welke bacterie de bron was voor een infectie onder verschillende patiënten en hoe flexibele endoscopen beter gedesinfecteerd kunnen worden. Een goede droogprocedure na de desinfectiefase blijkt hierbij van belang te zijn. Kovaleva gaat in op infecties en kruisbesmettingen die verband houden met endoscopische procedures. Besmetting en infectie kunnen samenhangen met het niet effectief kunnen verwijderen van bacteriën in de biofilm in de endoscoop. Kovaleva ontwikkelde een in vitro model om de effecten van desinfectie met perazijnzuur en drogen met verwarmde lucht te testen op biofilmvorming door micro-organismen die ze isoleerde uit besmette endoscopen. De hoge effectiviteit en het belang van een droogprocedure na een desinfectiefase van flexibele endoscopen tegen verschillende micro-organismen in biofilms werd in deze studie aangetoond. Biofilmvorming in een endoscoop kan leiden tot het falen van de desinfectie en besmetting en infecties bij patiënten die worden behandeld met flexibele endoscopen met een biofilm. Microbiologische monitoring van het hergebruik van flexibele endoscopen is geschikt om besmettingen op te sporen en infecties te voorkomen of te beperken bij patiënten na endoscopische procedures.

Patiënten met een parodontale aandoening reageren zo verschillend op antibiotica, dat een microbiologische analyse uitkomst kan bieden. Die kan laten zien welke antibioticum het probleem aanpakt, en welke niet. Dat concludeert Thomas Rams in een onderzoek in de USA naar het effect van antibiotica bij behandelingen van parodontitis en infecties rond implantaties. Antibiotica spelen een sleutelrol in het behandelen van parodontitis of vergelijkbare infecties rondom tandheelkundige implantaten. Rams vergeleek in een aantal studies de effecten van specifieke antibiotica op specifieke bacteriën (Streptococcus constellatus, Streptococcus intermedius en Enterococcus faecalis) Hij concludeert dat combinaties van antibiotica meer effect hebben op parodontale aandoeningen dan hoge concentraties van één antibioticum. Ook stelt hij dat van patiënt tot patiënt verschilt hoe het geïnfecteerde gebied reageert op antibiotica. Voor een effectieve behandeling raadt hij daarom een microbiologische analyse aan, voorafgaand aan het starten met antibiotica. Zo’n analyse kan in kaart brengen welk antibioticum de infectie kan aanpakken. De uitkomsten kunnen helpen om de zorg aan patiënten met chronische parodontitis en geïnfecteerde implantaten te verbeteren.

Als een endoscoop niet goed schoongemaakt wordt bestaat de kans dat infecties van patiënt op patiënt worden overgedragen. UMCG-onderzoeker Julia Kovaleva bestudeerde welke bacterie de bron was voor een infectie onder verschillende patiënten en hoe flexibele endoscopen beter gedesinfecteerd kunnen worden. Een goede droogprocedure na de desinfectiefase blijkt hierbij van belang te zijn. Kovaleva gaat in op infecties en kruisbesmettingen die verband houden met endoscopische procedures. Besmetting en infectie kunnen samenhangen met het niet effectief kunnen verwijderen van bacteriën in de biofilm in de endoscoop. Kovaleva ontwikkelde een in vitro model om de effecten van desinfectie met perazijnzuur en drogen met verwarmde lucht te testen op biofilmvorming door micro-organismen die ze isoleerde uit besmette endoscopen. De hoge effectiviteit en het belang van een droogprocedure na een desinfectiefase van flexibele endoscopen tegen verschillende micro-organismen in biofilms werd in deze studie aangetoond. Biofilmvorming in een endoscoop kan leiden tot het falen van de desinfectie en besmetting en infecties bij patiënten die worden behandeld met flexibele endoscopen met een biofilm. Microbiologische monitoring van het hergebruik van flexibele endoscopen is geschikt om besmettingen op te sporen en infecties te voorkomen of te beperken bij patiënten na endoscopische procedures.

Als een endoscoop niet goed schoongemaakt wordt bestaat de kans dat infecties van patiënt op patiënt worden overgedragen. UMCG-onderzoeker Julia Kovaleva bestudeerde welke bacterie de bron was voor een infectie onder verschillende patiënten en hoe flexibele endoscopen beter gedesinfecteerd kunnen worden. Een goede droogprocedure na de desinfectiefase blijkt hierbij van belang te zijn. Kovaleva gaat in op infecties en kruisbesmettingen die verband houden met endoscopische procedures. Besmetting en infectie kunnen samenhangen met het niet effectief kunnen verwijderen van bacteriën in de biofilm in de endoscoop. Kovaleva ontwikkelde een in vitro model om de effecten van desinfectie met perazijnzuur en drogen met verwarmde lucht te testen op biofilmvorming door micro-organismen die ze isoleerde uit besmette endoscopen. De hoge effectiviteit en het belang van een droogprocedure na een desinfectiefase van flexibele endoscopen tegen verschillende micro-organismen in biofilms werd in deze studie aangetoond. Biofilmvorming in een endoscoop kan leiden tot het falen van de desinfectie en besmetting en infecties bij patiënten die worden behandeld met flexibele endoscopen met een biofilm. Microbiologische monitoring van het hergebruik van flexibele endoscopen is geschikt om besmettingen op te sporen en infecties te voorkomen of te beperken bij patiënten na endoscopische procedures.

Patiënten met een parodontale aandoening reageren zo verschillend op antibiotica, dat een microbiologische analyse uitkomst kan bieden. Die kan laten zien welke antibioticum het probleem aanpakt, en welke niet. Dat concludeert Thomas Rams in een onderzoek in de USA naar het effect van antibiotica bij behandelingen van parodontitis en infecties rond implantaties. Antibiotica spelen een sleutelrol in het behandelen van parodontitis of vergelijkbare infecties rondom tandheelkundige implantaten. Rams vergeleek in een aantal studies de effecten van specifieke antibiotica op specifieke bacteriën (Streptococcus constellatus, Streptococcus intermedius en Enterococcus faecalis) Hij concludeert dat combinaties van antibiotica meer effect hebben op parodontale aandoeningen dan hoge concentraties van één antibioticum. Ook stelt hij dat van patiënt tot patiënt verschilt hoe het geïnfecteerde gebied reageert op antibiotica. Voor een effectieve behandeling raadt hij daarom een microbiologische analyse aan, voorafgaand aan het starten met antibiotica. Zo’n analyse kan in kaart brengen welk antibioticum de infectie kan aanpakken. De uitkomsten kunnen helpen om de zorg aan patiënten met chronische parodontitis en geïnfecteerde implantaten te verbeteren.

Bij de routinematige controle van sterilisatie en hygienebeheersing in de Nederlandse destructoren is in het onderzoekjaar 1984 gebleken dat in 10% van de monsters uit bedrijfsruimten nog Salmonella kon worden geisoleerd, doch dat deze bacteriesoort uit geen enkel monster gesteriliseerd eindprodukt kon worden gekweekt. De Enterobacteriaceae kiemgetallen van monsters uit bedrijfsruimten en van eindprodukten waren in de meeste gevallen zeer laag, hetgeen op een in het algemeen goede hygiene in de bedrijven wijst. De gevolgde sterilisatieprocedures bleken niet altijd even effectief. In 15 tot 28% van de onderzochte monsters konden nog bacteriesporen worden aangetoond. In de nabije toekomst zal extra aandacht aan dit probleem worden besteed, omdat het van het grootste belang is dat eventueel aanwezige ziektekiemen in de destructiematerialen volledig worden geelimineerd, alvorens tot hergebruik van deze materialen wordt overgegaan.

Health complaints may occur after swimming in feacally polluted surface water that complies with the microbial water quality standards. To give the Dutch standards a scientific base and to determine whether existing standards should be made more stringent, research into the relationship between microbial water quality and the occurrence of health complaints should be carried out. To explore the merit and feasibility of such an investigation among endurance competitions, a feasibility study was undertaken among 314 participants of the quarter triathlon in Ammerstol (the water exposed population) and 81 participants of the Run-Bike-Run competition in Borne (the control population), both held at August 29th 1992. Information on the occurrence of health complaints during and in the week after the competition day was obtained by the use of a postal questionnaire. On the competition day the swimming water of the triathlon was sampled to examine the microbial quality. The results showed that the triathletes and Run-Bike-Run athletes were comparable with respect to other factors except exposure to water that may influence the occurrence of health complaints. In the week after the race health complaints (e.g. gastrointestinal symptoms, respiratory symptoms and skin/mucosal complaints) were reported by 14,8% of the triathletes and 7,4% of the Run-Bike-Run athletes. At the time of the triathlon the microbial quality of the swimming water complied with the Dutch standards. It was concluded that for studying the relationship between microbial water quality and health complaints a study among triathletes and Run-Bike-Run athletes is feasible. This study will be carried out in the summer of 1993.

Bij het regelmatig onderzoek van de 4 Nederlandse destructoren naar het voorkomen van Enterobact. en Salmonella is gebleken dat de algemene hygiene in deze bedrijven op hetzelfde niveau als andere jaren is gebleven. Wat Salmonella betreft wordt een lichte stijging in het percentage positieve monsters uit de bedrijfsruimten geconstateerd en een geringe daling van het percentage positieve monsters van eindprodukten. In totaal bevatten nu 12,5% van de monsters uit de bedrijfsruimten Salmonella. Deze kiem kon in 2,5% van de gesteriliseerde eindprodukten worden teruggevonden. Er zijn aanwijzingen, dat in 3 van de 4 destructoren persisterende besmettingen met Salmonella zijn opgetreden, hetgeen zou duiden op onvoldoende reiniging en desinfectie. De besmetting met Enterobacteriacea van alle onderzochte monsters bleek over het algemeen gering, zodat mag worden geconcludeerd dat de gevonden Salmonella- besmettingen in de meeste gevallen uit kleine aantallen kiemen hebben bestaan. Blijvende aandacht zal nodig zijn voor het instandhouden van de hygiene.

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De milieuveiligheid van diverse gewasbeschermingsmiddelen van natuurlijke oorsprong zal de komende jaren door de EU opnieuw worden beoordeeld. Bovendien zullen nieuwe middelen op de markt komen vanwege het toenemende belang van duurzame landbouw. Er zijn echter nauwelijks richtlijnen om de milieuveiligheid van dergelijke middelen te evalueren. Daarom worden in deze bureaustudie twee hulpmiddelen aangereikt om de milieuveiligheid van natuurlijke gewasbeschermingsmiddelen beter te kunnen beoordelen. Het eerste betreft beslisbomen om voor drie groepen onacceptabele potentiele risico's te kunnen scheiden van acceptabele. De groepen zijn microbiele gewasbeschermingsmiddelen, signaalstoffen als feromonen en middelen van plantaardige oorsprong. Het tweede hulpmiddel bestaat uit "samenvattings"-tabellen als format om de uitkomsten van experimenten met microbiele gewasbeschermingsmiddelen en de betrouwbaarheid en bruikbaarheid daarvan voor veiligheidsevaluaties vast te leggen. Van nature voorkomende gewasbeschermingsmiddelen zijn doorgaans minder persistent en toxisch dan synthetische gewasbeschermingsmiddelen. Het is echter niet juist om middelen van natuurlijke oorsprong derhalve te vrijwaren van milieuveiligheidsevaluaties. Ze kunnen infecties of vergiftigingen veroorzaken afhankelijk van de microbiele of chemische eigenschappen, de dosering, het type en de plaats van toepassing. Het is van het eerste belang de identiteit van het micro-organisme, de stof of het mengsel van stoffen, de microbiele of (bio)chemische eigenschappen, het werkingsmechanisme, de oorsprong, de eventuele rol onder natuurlijke omstandigheden en het gastheerbereik vast te stellen, indien van toepassing. Vervolgens kan, mede op grond van relevante wetenschappelijke literatuur en/of speciaal daartoe uitgevoerde experimenten, worden vastgesteld in hoeverre het gebruik van deze middelen veilig voor het milieu is.

niet beschikbaar

Bij het regelmatig onderzoek van de 4 Nederlandse destructoren naar het voorkomen van Enterobacteriaceae en Salmonella, als maat voor de algemene hygiene in bedrijven, is gebleken dat in 1982 geen belangrijke verschuivingen zijn opgetreden in vergelijking tot 1981. Het meest opmerkelijke is dat de aanzienlijke Salmonellabesmetting in de bedrijfsruimten, zoals geconstateerd in 1981, dit jaar weer is afgenomen. In totaal bevatte nu 10% van de monsters uit de bedrijfsruimten Salmonella. Deze kiem kon in 4% van de gesteriliseerde eindprodukten worden teruggevonden. De besmetting met Enterobact. van alle onderzochte monsters bleek over het algemeen gering, zodat mag worden geconcludeerd dat de gevonden Salmonella-besmettingen in de meeste gevallen uit kleine aantallen kiemen hebben bestaan. Blijvende aandacht zal nodig zijn voor het instandhouden van de hygiene in de bedrijfsruimten. Ook dit jaar is ws. in 1 van de bedrijven een langdurige besmetting van bedrijfsruimten aangetroffen, welke tot contaminatie van eindprodukt. aanleiding heeft gegeven.