Staff Publications

Staff Publications

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    'Staff publications' is the digital repository of Wageningen University & Research

    'Staff publications' contains references to publications authored by Wageningen University staff from 1976 onward.

    Publications authored by the staff of the Research Institutes are available from 1995 onwards.

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Bacillus cereus growth and biofilm formation: the impact of substratum, iron sources, and transcriptional regulator Sigma 54
Hayrapetyan, Hasmik - \ 2017
University. Promotor(en): Tjakko Abee, co-promotor(en): Masja Nierop Groot. - Wageningen : Wageningen University - ISBN 9789463431194 - 181
microorganisms - bacillus cereus - food contamination - biofilms - foodborne pathogens - abiotic conditions - sporulation - micro-organismen - voedselbesmetting - voedselpathogenen - abiotiek - sporulatie

Biofilms are surface-associated communities of microbial cells embedded in a matrix of extracellular polymers. It is generally accepted that the biofilm growth mode represents the most common lifestyle of microorganisms. Next to beneficial biofilms used in biotechnology applications, undesired biofilms can be formed by spoilage and pathogenic microorganisms in food production environments. Bacillus cereus is a foodborne human pathogen able to cause two types of food poisoning, emetic and diarrheal. B. cereus can persist in factory environments in the form of biofilms, which can become a source of food contamination. This thesis adds to the knowledge about (a)biotic factors and conditions that affect B. cereus biofilm formation, including the effect of type of substratum such as polystyrene and stainless steel, with the latter supporting the highest biofilm formation for all tested strains including two reference strains and 20 food isolates. The ability of B. cereus to use a variety of iron sources was subsequently studied in these 22 strains and linked to the genes encoding iron transport systems present in the respective genomes, revealing significant diversity in the capacity to use complex and non-complex iron sources for growth and biofilm formation. For spore forming Bacilli, biofilm formation and sporulation are two intertwined cellular processes and studies in wet and dry (air-exposed) biofilms revealed differences in sporulation rate and efficacy, with biofilm-derived spores showing higher heat resistance than their planktonic counterparts. Additionally, comparative phenotype and transcriptome analysis of B. cereus wild type and a Sigma 54 deletion mutant provided insight into the pleiotropic role of this transcriptional regulator in B. cereus biofilm formation and physiology in general. Taken together, this knowledge improves our understanding of the biofilm lifecycle of this notorious food-borne human pathogen and provides clues which can help to reduce the domestication of this microorganism in production environments.

Characterisation of Lactobacillus plantarum single and multi-strain biofilms
Fernández Ramírez, Mónica D. - \ 2016
University. Promotor(en): Tjakko Abee; Eddy Smid, co-promotor(en): Masja Nierop Groot. - Wageningen : Wageningen University - ISBN 9789462579217 - 169
lactobacillus plantarum - biofilms - food spoilage - models - environmental factors - voedselbederf - modellen - milieufactoren

Biofilms consist of microorganisms attached to a surface and embedded in a protective matrix of extracellular polymeric substances. Within a biofilm, micro-organisms are protected from harsh environmental conditions including those resulting from cleaning and disinfecting agents leading to food safety and quality issues after dispersal of life biofilm cells and subsequent contamination of foods. In this thesis, single and multi-strain biofilm formation by Lactobacillus plantarum isolates was characterised including the model strain L. plantarum WCFS1 and food spoilage isolates. Analysis of the L. plantarum single strain biofilm formation showed a role for proteins and/or proteinaceous material in surface colonization and extracellular DNA as components of the biofilm matrix. The relevance of lysis for the build-up of the biofilm matrix with eDNA was demonstrated using L. plantarum WCFS1 mutants affected in the production of cell wall polysaccharides resulting in altered cell surface composition and mutants lacking cell wall lytic enzymes that showed decreased cell lysis. Dual and multi-strain biofilms were studied using quantitative PCR and next generation sequencing based on detection of strain specific alleles in competitive planktonic and surface-attached biofilm growth models. In multi-strain cultures, the performance of individual strains generally correlated with their performance in pure culture, and relative strain abundance in multi-strain static biofilms positively correlated with the relative strain abundance in suspended (planktonic) cultures. Performance of individual strains in dual-strain biofilms was highly influenced by the presence of the secondary strain, and in most cases no correlation between the relative contributions of viable planktonic cells and viable cells in the biofilm was noted. The next generation sequencing approach provided additional insights in the performance of twelve individual L. plantarum strains in static and dynamic flow competitive biofilm models and showed that environmental stresses such as absence of Mn(II) and increased temperature affected not only the relative abundance of each strain both in planktonic and static biofilm growth but also the release of eDNA. The strains dominating the biofilms in static conditions were not the same as those dominating in biofilms developed in dynamic flowing conditions. The genome content of the dominating strains was explored to identify genetic factors that potentially contribute to strain specific competitive-biofilm forming capacity under dynamic flowing conditions, providing leads for further research. All the single, dual and multi-strain biofilms contained a considerable number of viable L. plantarum cells, representing a potential source of contamination. The developed tools and insights obtained in L. plantarum biofilm formation capacity may assist development of strategies to prevent (re)contamination from biofilms in food processing environments.

Microalgae production in a biofilm photobioreactor
Blanken, Ward - \ 2016
University. Promotor(en): Rene Wijffels, co-promotor(en): Marcel Janssen. - Wageningen : Wageningen University - ISBN 9789462578425 - 234
algae - algae culture - biofilms - bioreactors - growth - production costs - biomass - artificial lighting - photosynthesis - carbon dioxide - algen - algenteelt - bioreactoren - groei - productiekosten - biomassa - kunstmatige verlichting - fotosynthese - kooldioxide

Microalgae can be used to produce high-value compounds, such as pigments or high value fatty acids, or as a feedstock for lower value products such as food and feed compounds, biochemicals, and biofuels. In order to produce these bulk products competitively, it is required to lower microalgae production cost. Production costs could be reduced by employing microalgae biofilms as a production platform. The main advantages of microalgae biofilms are a direct harvest of concentrated microalgae paste, and the uncoupling of the hydraulic retention time from the microalgal retention time. The latter allows to decrease the liquid volume or to employ dilute waste streams. To successfully employ biofilms, however, it is required that microalgal biofilms can be cultivated at high productivity and high photosynthetic efficiency. The aim of this thesis was to optimize the productivity of microalgal biofilms.

Light energy drives microalgal growth. Sunlight is free and abundant, but sunlight intensity varies over the day and the seasons. This makes it impossible to maintain optimal production conditions throughout the day. These fluctuations in irradiance can be prevented by applying artificial lighting. Although, artificial lighting will supply a constant light intensity and thus increase productivity and simplify process control, it will also increase microalgae production cost. A quantitative evaluation of lighting costs and energy requirement was still missing and this was the topic of Chapter 2. The costs related to artificial lighting were identified as 25.3 $ per kilogram of dry-weight biomass, with only 4% to 6% of the electrical energy required to power the lamps eventually stored as chemical energy in microalgal biomass. Energy loss and increased production cost may be acceptable for the production of high value products, but in general they should be avoided.

In Chapter 3, a photobioreactor design based on a rotating biological contactor (RBC) was introduced and used as a production platform for microalgal biomass cultivated in a biofilm. In the photobioreactor, referred to as the Algadisk, microalgae grow in biofilm on vertical rotating disks partially submerged in water with dissolved nutrients. The objective was to evaluate the potential of the Algadisk photobioreactor, and identify the window of operation of the process with respect to the effects of disk roughness, disk rotation speed and CO2 concentration. These parameters were evaluated in relation to biomass productivity, photosynthetic efficiency, and the long-term cultivation stability of the production process.

The mesophilic green microalga Chlorella sorokiniana was used as a model organism. In the lab-scale Algadisk reactor, a productivity of 20.1 ±0.7 gram per m2 disk surface per day and a biomass yield on light of 0.9 ±0.04 gram dry weight biomass per mol photons were obtained. This productivity could be retained over 21 weeks without re-inoculation. To obtain maximal and stable productivity it was important that the disk surface provides a structure that allows biomass retention on the disk after harvest. The retained biomass acts as inoculum for the new biofilm and is therefore essential for quick biofilm regrowth. Most important process parameters were CO2­ supply, temperature, and pH. Although deviations of these parameters from the optimal conditions resulted in productivity loss, the system quickly recovered when optimal conditions were restored. These results exhibit an apparent opportunity to employ the Algadisk photobioreactor and biofilm systems in general at large scale for microalgae biomass production provided CO2 supply is adequate.

In order to better understand the process conditions inside the biofilm a model was developed in the further chapters. These mathematical models were calibrated and validated with dedicated experiments. In Chapter 4 first a general applicable kinetic model was developed able to predict light limited microalgal growth. This model combines a mathematical description for photoautotrophic sugar production with a description for aerobic chemoheterotrophic biomass growth. The model is based on five measurable biological parameters which were obtained from literature for the purpose of this study. The model was validated on experiments described in literature for both Chlorella sorokiniana and Chlamydomonas reinhardtii. The specific growth rate was initially predicted with a low accuracy, which was most likely caused by simplifications in the light model and inaccurate parameter estimations. When optimizing the light model and input parameters the model accuracy was improved and validated. With this model a reliable engineering tool became available to predict microalgal growth in photobioreactors. This microalgal growth model was included in the biofilm growth models introduced in Chapters 5 and 6.

In Chapter 5 microalgal biofilms of Chlorella sorokiniana were grown under simulated day-night cycles at high productivity and high photosynthetic efficiency. The experimental data under day/night cycles were used to validate a microalgal biofilm growth model. For this purpose the light limited microalgal growth model from Chapter 4 was extended to include diurnal carbon-partitioning and maintenance under prolonged dark conditions. This new biofilm growth model was then calibrated and validated experimentally. Based on these experiments and model simulations no differences in the light utilization efficiency between diurnal and continuous light conditions were identified. Indirectly this shows that biomass lost overnight represents sugar consumption for synthesis of new functional biomass and maintenance related respiration. This is advantageous, as this result shows that it is possible to cultivate microalgae at high photosynthetic efficiencies on sunlight and that the night does not negatively impact overall daily productivity. Long periods of darkness resulted in reduced maintenance related respiration.

Based on simulations with the validated biofilm growth model it could be determined that the photosynthetic efficiency of biofilm growth is higher than that of suspension growth. This is related to the fact that the maintenance rate in the dark zones of the biofilm is lower compared to that in the dark zones of suspension cultures, which are continuously mixed with the photic zone.

In Chapter 3 it was identified that concentrated CO2 streams are required to obtain high productivities. However, over-supplying CO2 results into loss of CO2 to the environment and is undesirable for both environmental and economic reasons. In Chapter 6 the phototrophic biofilm growth model from Chapter 5 was extended to include CO2 and O2 consumption, production, and diffusion. The extended model was validated in growth experiments with CO2 as limiting substrate. Based on the validated model the CO2 utilization and productivity in biofilm photobioreactors were optimized by changing the gas flow rate, the number of biofilm reactors in series, and the gas composition. This resulted in a maximum CO2 utilization efficiency of 96% by employing flue gas, while the productivity only dropped 2% compared to non-CO2 limited growth. In order to achieve this 25 biofilm reactors units, or more, must be operated in series. Based on these results we conclude that concentrated CO2 streams and plug flow behaviour of the gaseous phase over the biofilm surface are essential for high CO2 utilization efficiencies and high biofilm productivity.

In Chapter 7 the implications of these studies for the further development of biofilm photobioreactors was discussed in the light of current biofilm photobioreactor designs. Design elements of state of the art biofilm photobioreactors, were combined into a new conceptual biofilm photobioreactor design. This new design combines all advantages of phototrophic biofilms minimizing the amount of material required. Further improvements by means of process control strategies were suggested that aim for maximal productivity and maximal nutrient utilization efficiency. These strategies include: control of the biofilm thickness, control of the temperature, and optimized nutrient supply strategies.

Fluidized Capacitive Bioanode As a Novel Reactor Concept for the Microbial Fuel Cell
Deeke, A. ; Sleutels, T.H.J.A. ; Donkers, T.F.W. ; Hamelers, B. ; Buisman, C.J.N. ; Heijne, A. ter - \ 2015
Environmental Science and Technology 49 (2015)3. - ISSN 0013-936X - p. 1929 - 1935.
waste-water treatment - electricity-generation - power-generation - iron reduction - scaled-up - performance - carbon - resistance - membranes - biofilms
The use of granular electrodes in Microbial Fuel Cells (MFCs) is attractive because granules provide a cost-effective way to create a high electrode surface area, which is essential to achieve high current and power densities. Here, we show a novel reactor design based on capacitive granules: the fluidized capacitive bioanode. Activated carbon (AC) granules are colonized by electrochemically active microorganisms, which extract electrons from acetate and store the electrons in the granule. Electricity is harvested from the AC granules in an external discharge cell. We show a proof-of-principle of the fluidized capacitive system with a total anode volume of 2 L. After a start-up period of 100 days, the current increased from 0.56 A/m2 with 100 g AC granules, to 0.99 A/m2 with 150 g AC granules, to 1.3 A/m2 with 200 g AC granules. Contact between moving AC granules and current collector was confirmed in a control experiment without biofilm. Contribution of an electro-active biofilm to the current density with recirculation of AC granules was limited. SEM images confirmed that a biofilm was present on the AC granules after operation in the fluidized capacitive system. Although current densities reported here need further improvement, the high surface area of the AC granules in combination with external discharge offers new and promising opportunities for scaling up MFCs.
Electricity generation by a plant microbial fuel cell with an integrated oxygen reducing biocathode
Wetser, K. ; Sudirjo, E. ; Buisman, C.J.N. ; Strik, D.P.B.T.B. - \ 2015
Applied energy 137 (2015). - ISSN 0306-2619 - p. 151 - 157.
determine performance - living plants - reduction - oxidation - biofilms - system
In this study we show that a chemical ferricyanide cathode can be replaced by a biological oxygen reducing cathode in a plant microbial fuel cell (PMFC) with a new record power output. A biocathode was successfully integrated in a PMFC and operated for 151 days. Plants growth continued and the power density increased reaching a maximum power output of 679 mW/m2 plant growth area (PGA) in a 10 min polarization. The two week record average power densities was 240 mW/m2 PGA. The new records were reached due to the high redox potential of oxygen reduction which was effectively catalyzed by microorganisms in the cathode. This resulted in a 127 mV higher cathode potential of the PMFC with a biocathode than a PMFC with a ferricyanide cathode. We also found that substrate availability in the anode likely limits the current generation. This work is crucial for PMFC application as it shows that PMFC can be a completely sustainable biotechnology with an improved power output.
Protecting with nature (PwN) PwN concept (bio-) corrosion prevention
Mijle Meijer, H. van der; Foekema, E.M. ; Leon, F. - \ 2014
Delft : TNO (TNO report TNO 2014 R11887) - 18
corrosiebescherming - staal - biofilms - mariene gebieden - havens - mariene constructies - innovaties - duurzaamheid (sustainability) - corrosion protection - steel - marine areas - harbours - marine structures - innovations - sustainability
Harbour infrastructures, civil engineering structures and offshore structures are exposed to a very aggressive maritime environment. The local corrosion mechanism bio-corrosion or microbial influenced corrosion (MIC) seems to be the life determining failure mechanism for these structures. There is a need for durable solutions to protect and maintain these structures. Different corrosion protection methods and systems are commercially available but each application requires a specific protection system. The optimal solution is not available yet
Vleeskuikenhouders: controleer regelmatig uw drinkwaterkwaliteit!
Harn, J. van; Neijenhuis, F. ; Ellen, H.H. - \ 2014
pluimveehouderij - vleeskuikens - drinkwater - waterkwaliteit - biofilms - diergezondheid - poultry farming - broilers - drinking water - water quality - animal health
In deze brochure staan achtergronden, aandachtspunten en tips voor goed en gezond drinkwater voor vleeskuikens.
Inactivation of Listeria monocytogenes by disinfectants and bacteriophages in suspension and stainless steel carrier tests
Chaitiemwong, N. ; Hazeleger, W.C. ; Beumer, R.R. - \ 2014
Journal of Food Protection 77 (2014)12. - ISSN 0362-028X - p. 2012 - 2020.
quaternary ammonium compound - food-contact surfaces - salmonella-typhimurium - processing equipment - cross-contamination - planktonic cells - biofilms - bacteria - efficacy - infection
To simulate food contact surfaces with pits or cracks, stainless steel plates with grooves (depths between 0.2 and 5 mm) were constructed. These plates were artificially contaminated with Listeria monocytogenes in clean conditions, with organic soiling, or after 14 days of biofilm formation after which inactivation of the pathogen by Suma Tab D4 (sodium dichloroisocyanurate, 240 and 300 mg/liter), Suma Bac D10 (quaternary ammonium compound, 740 mg/liter), and bacteriophage suspension (Listex P100) was determined. Both chemical disinfectants performed well in suspension tests and in clean carrier tests according to the European standard with a reduction of more than 5 and 4 log units, respectively, of Listeria cells after 5 min of contact time. However, for the plates with grooves, the reduction could not meet the standard requirement, although a higher reduction of L. monocytogenes was observed in the shallow grooves compared with the deeper grooves. Furthermore, presence of food residues and biofilm reduced the effect of the disinfectants especially in the deep grooves, which was dependent on type of food substrate. Bacteriophages showed the best antimicrobial effect compared with the chemical disinfectants (sodium dichloroisocyanurate and quaternary ammonium compound) in most cases in the shallow grooves, but not in the deep grooves. The chlorine based disinfectants were usually less effective than quaternary ammonium compound. The results clearly demonstrate that surfaces with grooves influenced the antimicrobial effect of the chemical disinfectants and bacteriophages because the pathogen is protected in the deep grooves. The use of bacteriophages to inactivate pathogens on surfaces could be helpful in limited cases; however, use of large quantities in practice may be costly and phage-resistant strains may develop.
Nanoscale cell wall deformation impacts long-range bacterial adhesion forces on surfaces
Chen, Y. ; Harapanahalli, A.K. ; Busscher, H.J. ; Norde, W. ; Mei, H.C. van der - \ 2014
Applied and Environmental Microbiology 80 (2014)2. - ISSN 0099-2240 - p. 637 - 643.
staphylococcus-aureus - microscopy - biofilms - attraction - mechanisms - dlvo - van
Adhesion of bacteria occurs on virtually all natural and synthetic surfaces and is crucial for their survival. Once they are adhering, bacteria start growing and form a biofilm, in which they are protected against environmental attacks. Bacterial adhesion to surfaces is mediated by a combination of different short- and long-range forces. Here we present a new atomic force microscopy (AFM)-based method to derive long-range bacterial adhesion forces from the dependence of bacterial adhesion forces on the loading force, as applied during the use of AFM. The long-range adhesion forces of wild-type Staphylococcus aureus parent strains (0.5 and 0.8 nN) amounted to only one-third of these forces measured for their more deformable isogenic ¿pbp4 mutants that were deficient in peptidoglycan cross-linking. The measured long-range Lifshitz-Van der Waals adhesion forces matched those calculated from published Hamaker constants, provided that a 40% ellipsoidal deformation of the bacterial cell wall was assumed for the ¿pbp4 mutants. Direct imaging of adhering staphylococci using the AFM peak force-quantitative nanomechanical property mapping imaging mode confirmed a height reduction due to deformation in the ¿pbp4 mutants of 100 to 200 nm. Across naturally occurring bacterial strains, long-range forces do not vary to the extent observed here for the ¿pbp4 mutants. Importantly, however, extrapolating from the results of this study, it can be concluded that long-range bacterial adhesion forces are determined not only by the composition and structure of the bacterial cell surface but also by a hitherto neglected, small deformation of the bacterial cell wall, facilitating an increase in contact area and, therewith, in adhesion force.
High loaded MBRs for organic matter recovery from sewage: Effect of solids retention time on bioflocculation and on the role of extracellular polymers
Faust, L. ; Temmink, B.G. ; Zwijnenburg, A. ; Kemperman, A.J.B. ; Rijnaarts, H. - \ 2014
Water Research 56 (2014). - ISSN 0043-1354 - p. 258 - 266.
waterzuivering - membranen - biofilms - organische stof - water treatment - membranes - organic matter - municipal waste-water - submerged membrane bioreactor - improved energy recovery - activated-sludge process - microbial community - surface-properties - substances eps - performance - extraction - constituents
High loaded MBRs (HL-MBR) can concentrate sewage organic matter by aerobic bioflocculation for subsequent anaerobic conversion to methane or volatile fatty acids. In the range of very short solid retention times (SRT), the effect of SRT on bioflocculation and EPS production in HL-MBR was investigated. This short SRT range was selected to find an optimum SRT maximising recovery of organics by aerobic bioflocculation and minimizing losses of organics by aerobic mineralization. Bioflocculation was studied in five HL-MBRs operated at SRTs of 0.125, 0.25, 0.5, 1 and 5 d. The extent of flocculation, defined as the fraction of suspended COD in the concentrate, increased from 59% at an SRT of 0.125 d to 98% at an SRT of 5 d. The loss of sewage organic matter by biological oxidation was 1, 2, 4, 11 and 32% at SRT of 0.125–5 d. An SRT of 0.5–1 d gave best combination of bioflocculation and organic matter recovery. Bound extracellular polymeric substances (EPS) concentrations, in particular EPS-protein concentrations, increased when the SRT was prolonged from 0.125 to 1 d. This suggests that these EPS-proteins govern the bioflocculation process. A redistribution took place from free (supernatant) EPS to bound (floc associated) EPS when the SRT was prolonged from 0.125 to 1 d, further supporting the fact that the EPS play a dominant role in the flocculation process. Membrane fouling was most severe at the shortest SRTs of 0.125 d. No positive correlation was detected between the concentration of free EPS and membrane fouling, but the concentration of submicron (45–450 nm) particles proved to be a good indicator for this fouling.
Balancing the organic load and light supply in symbiotic microalgal–bacterial biofilm reactors treating synthetic municipal wastewater
Boelee, N.C. ; Temmink, B.G. ; Janssen, M. ; Buisman, C.J.N. ; Wijffels, R.H. - \ 2014
Ecological Engineering 64 (2014). - ISSN 0925-8574 - p. 213 - 221.
afvalwaterbehandeling - biofilms - symbiose - algen - bacteriën - heterotrofe micro-organismen - fotosynthese - acetaten - stikstof - fosfor - nitrificatie - denitrificatie - biologische waterzuiveringsinstallaties - biobased economy - waste water treatment - symbiosis - algae - bacteria - heterotrophic microorganisms - photosynthesis - acetates - nitrogen - phosphorus - nitrification - denitrification - biological water treatment plants - activated-sludge - nutrient removal - growth - phytoplankton - fluorescence - enhancement
Symbiotic microalgal–bacterial biofilms can be very attractive for municipal wastewater treatment. Microalgae remove nitrogen and phosphorus and simultaneously produce the oxygen that is required for the aerobic, heterotrophic degradation of organic pollutants. For the application of these biofilms in new wastewater treatment systems, the engineering aspects need to be investigated to obtain a balanced system where no additional oxygen is required. In this study symbiotic microalgal–bacterial biofilms were grown in flow cells with ammonium and phosphate, and with acetate as biodegradable organic pollutant at a hydraulic retention time of 4.5 h. The symbiotic biofilms removed acetate from 323 mg/L to 39 mg/L without an external oxygen or carbon dioxide supply at a removal rate of 43 g COD/m2/d. Ammonium and phosphate could not be completely removed, but removal rates of 3.2 g/m2/d and 0.41 g/m2/d were obtained, respectively. Further nitrogen removal may be obtained by nitrification and denitrification as the biofilm obtained a considerable heterotrophic denitrification capacity. The symbiotic relationship between microalgae and aerobic heterotrophs was proven by subsequently removing light and acetate. In both cases this resulted in the cessation of the symbiosis and in increasing effluent concentrations of both acetate and the nutrients ammonium and phosphate. Future research should investigate the dimensioning of an up-scaled symbiotic biofilm reactor, and the possibilities to obtain additional nitrogen and phosphorus removal under day–night cycles utilizing real wastewater.
Nutrient Removal and Biomass Production in an Outdoor Pilot-Scale Phototrophic Biofilm Reactor for Effluent Polishing
Boelee, N.C. ; Janssen, M. ; Temmink, H. ; Shrestha, R. ; Buisman, C.J.N. ; Wijffels, R.H. - \ 2014
Applied Biochemistry and Biotechnology 172 (2014)1. - ISSN 0273-2289 - p. 405 - 422.
afvalwaterbehandeling - biofilms - algen - biologische waterzuiveringsinstallaties - fototropie - nitraten - fosfaten - waste water treatment - algae - biological water treatment plants - phototropism - nitrates - phosphates - waste-water treatment - rate algal pond - marine-phytoplankton - seasonal succession - phosphate-uptake - nitrogen uptake - nitrate uptake - phosphorus - growth - light
An innovative pilot-scale phototrophic biofilm reactor was evaluated over a 5-month period to determine its capacity to remove nitrogen and phosphorus from Dutch municipal wastewater effluents. The areal biomass production rate ranged between 2.7 and 4.5 g dry weight/m2/day. The areal nitrogen and phosphorus removal rates averaged 0.13 g N/m2/day and 0.023 g P/m2/day, which are low compared to removal rates achieved in laboratory biofilm reactors. Nutrient removal increased during the day, decreased with decreasing light intensity and no removal occurred during the night. Additional carbon dioxide supply was not requisite as the wastewater was comprised of enough inorganic carbon to sustain microalgal growth. The study was not conclusive for the limiting factor that caused the low nutrient removal rate, possibly the process was limited by light and temperature, in combination with pH increases above pH 9 during the daytime. This pilot-scale study demonstrated that the proposed phototrophic biofilm reactor is not a viable post-treatment of municipal wastewater effluents under Dutch climate conditions. However, the reactor performance may be improved when controlling the pH and the temperatures in the morning. With these adaptations, a phototrophic biofilm reactor could be feasible at lower latitudes with higher irradiance levels.
The effect of harvesting on biomass production and nutrient removal in phototrophic biofilm reactors for effluent polishing
Boelee, N.C. ; Janssen, M. ; Temmink, H. ; Taparaviciute, L. ; Khiewwijit, R. ; Janoska, A. ; Buisman, C.J.N. ; Wijffels, R.H. - \ 2014
Journal of Applied Phycology 26 (2014)3. - ISSN 0921-8971 - p. 1439 - 1452.
afvalwaterbehandeling - biofilms - dikte - dichtheid - algen - biologische waterzuiveringsinstallaties - fototropie - stikstof - fosfor - verwijdering - biobased economy - waste water treatment - thickness - density - algae - biological water treatment plants - phototropism - nitrogen - phosphorus - removal - waste-water treatment - photosynthetic efficiency - chlorella-sorokiniana - microalgal biofilms - phosphorus removal - mass-transport - fresh-water - light - growth
An increasing number of wastewater treatment plants require post-treatment to remove residual nitrogen and phosphorus. This study investigated various harvesting regimes that would achieve consistent low effluent concentrations of nitrogen and phosphorus in a phototrophic biofilm reactor. Experiments were performed in a vertical biofilm reactor under continuous artificial lighting and employing artificial wastewater. Under similar conditions, experiments were performed in near-horizontal flow lanes with biofilms of variable thickness. It was possible to maintain low nitrogen and phosphorus concentrations in the effluent of the vertical biofilm reactor by regularly harvesting half of the biofilm. The average areal biomass production rate achieved a 7 g dry weight m-2 day-1 for all different harvesting frequencies tested (every 2, 4, or 7 days), corresponding to the different biofilm thicknesses. Apparently, the biomass productivity is similar for a wide range of biofilm thicknesses. The biofilm could not be maintained for more than 2 weeks as, after this period, it spontaneously detached from the carrier material. Contrary to the expectations, the biomass production doubled when the biofilm thickness was increased from 130 µm to 2 mm. This increased production was explained by the lower density and looser structure of the 2 mm biofilm. It was concluded that, concerning biomass production and labor requirement, the optimum harvesting frequency is once per week.
Salmonella biofilms
Castelijn, G.A.A. - \ 2013
University. Promotor(en): Tjakko Abee; Marcel Zwietering, co-promotor(en): Roy Moezelaar. - S.l. : s.n. - ISBN 9789461737335 - 168
salmonella typhimurium - salmonella - biofilms - franjes - fimbriae

Biofilm formation by Salmonellaspp. is a problem in the food industry, since biofilms may act as a persistent source of product contamination. Therefore the aim of this study was to obtain more insight in the processes involved and the factors contributing to Salmonellabiofilm formation. A collection of SalmonellaTyphimurium clinical, outbreak-related and retail product isolates, was used to determine biofilm formation capacity and to identify cellular parameters contributing to surface colonisation. The results revealed dense biofilm formation by these isolates at 25 °C and 37 °C in nutrient-rich media. However, in nutrient-low media dense biofilm formation was only observed at 25 °C with industrial isolates. In addition, temperature and medium composition were also found to influence biofilm morphology and composition. At nutrient-low conditions at 25 °C the biofilm consisted of cell clusters encapsulated by an extracellular matrix composed of curli fimbriae and cellulose. In nutrient-rich conditions a monolayer of cells with little to no extracellular matrix were observed, with a prominent role for type 1 fimbriae. This type of fimbriae was only expressed in a subset of strains and appeared to contribute to initial attachment of Salmonellacells ultimately leading to dense biofilm formation. This study also indicated that biofilm formation differs between and within the Salmonellaserovars Typhimurium, Derby, Brandenburg and Infantis, isolated from meat processing environments. And, for all serovars biofim formation contributed to the survival on stainless steel surfaces and biofilm cells were less susceptible to peracetic acid disinfection treatments. This latter effect was specifically observed in the presence of organic matter, which drastically decreased the activity of peracetic acid conceivably resulting in low level exposure of the bacterial flora facilitating survival. Furthermore, single and repeated exposure to sub-lethal concentrations of the disinfectant benzalkonium chloride rapidly selected for resistant variants. In conclusion, the results obtained in this study may contribute to the development of better strategies for Salmonella control in food processing environments.

Microalgal biofilms for wastewater treatment
Boelee, N.C. - \ 2013
University. Promotor(en): Rene Wijffels, co-promotor(en): Hardy Temmink; Marcel Janssen. - [S.l. : S.n. - ISBN 9789461736666 - 213
algen - biofilms - afvalwaterbehandeling - bioproceskunde - milieutechnologie - algenteelt - mineralenopname - biomassa productie - biobased economy - algae - waste water treatment - bioprocess engineering - environmental technology - algae culture - mineral uptake - biomass production
The objective of this thesis was to explore the possibilities of using microalgal biofilms for the treatment of municipal wastewater, with a focus on the post-treatment of municipal wastewater effluent. The potential of microalgal biofilms for wastewater treatment was first investigated using a scenario analysis. Then biofilms were grown on wastewater treatment plant effluent in horizontal flow cells under different nutrient loads to determine the maximum uptake capacity of the biofilms for NO3 and PO4. Subsequently, microalgal biofilms were grown in a vertical laboratory-scale biofilm reactor. The effect of harvesting and biofilm thickness on the biomass production and nutrient removal was investigated. The biofilm reactor was taken outdoors and a vertical pilot-scale biofilm reactor was evaluated as post-treatment of municipal wastewater in a pilot-study. Finally, symbiotic microalgal-bacterial biofilms were investigated for full treatment of (pre-settled) wastewater.
Bacterial cell surface deformation under external loading
Chen, Y. ; Norde, W. ; Mei, H.C. van der; Busscher, H.J. - \ 2012
MBio 3 (2012)6. - ISSN 2150-7511 - 8 p.
atomic-force microscopy - staphylococcus-aureus - mechanical-properties - adhesion - biofilms - model - viscoelasticity - peptidoglycan - contact - removal
Viscoelastic deformation of the contact volume between adhering bacteria and substratum surfaces plays a role in their adhesion and detachment. Currently, there are no deformation models that account for the heterogeneous structure and composition of bacteria, consisting of a relatively soft outer layer and a more rigid, hard core enveloped by a cross-linked peptidoglycan layer. The aim of this paper is to present a new, simple model to derive the reduced Young’s modulus of the contact volume between adhering bacteria and substratum surfaces based on the relationship between deformation and applied external loading force, measured using atomic force microscopy. The model assumes that contact is established through a cylinder with constant volume and does not require assumptions on the properties and dimensions of the contact cylinder. The reduced Young’s moduli obtained (8 to 47 kPa) and dimensions of the contact cylinders could be interpreted on the basis of the cell surface features and cell wall characteristics, i.e., surfaces that are more rigid (because of either less fibrillation, less extracellular polymeric substance production, or a higher degree of cross-linking of the peptidoglycan layer) had shorter contact cylinders and higher reduced Young’s moduli. Application of an existing Hertz model to our experimental data yielded reduced Young’s moduli that were up to 100 times higher for all strains investigated, likely because the Hertz model pertains to a major extent to the more rigid peptidoglycan layer and not only to the soft outer bacterial cell surface, involved in the bond between a bacterium and a substratum surface.
Scenario Analysis of Nutrient Removal from Municipal Wastewater by Microalgal Biofilms
Boelee, N.C. ; Temmink, H. ; Janssen, M. ; Buisman, C.J.N. ; Wijffels, R.H. - \ 2012
Water 4 (2012)2. - ISSN 2073-4441 - p. 460 - 473.
afvalwaterbehandeling - biofilms - algen - biologische waterzuiveringsinstallaties - vergelijkend onderzoek - volgorden - haalbaarheidsstudies - heterotrofe micro-organismen - stikstof - fosfor - verwijdering - biomassa productie - biobased economy - waste water treatment - algae - biological water treatment plants - comparative research - sequences - feasibility studies - heterotrophic microorganisms - nitrogen - phosphorus - removal - biomass production - marine-phytoplankton - chemical-composition - chlorella-vulgaris - nitrate uptake - algal biofilm - growth - photobioreactor - photosynthesis
Microalgae can be used for the treatment of municipal wastewater. The application of microalgal biofilms in wastewater treatment systems seems attractive, being able to remove nitrogen, phosphorus and COD from wastewater at a short hydraulic retention time. This study therefore investigates the area requirement, achieved effluent concentrations and biomass production of a hypothetical large-scale microalgal biofilm system treating municipal wastewater. Three scenarios were defined: using microalgal biofilms: (1) as a post-treatment; (2) as a second stage of wastewater treatment, after a first stage in which COD is removed by activated sludge; and (3) in a symbiotic microalgal/heterotrophic system. The analysis showed that in the Netherlands, the area requirements for these three scenarios range from 0.32 to 2.1 m2 per person equivalent. Moreover, it was found that it was not possible to simultaneously remove all nitrogen and phosphorus from the wastewater, because of the nitrogen:phosphorus ratio in the wastewater. Phosphorus was limiting in the post-treatment scenario, while nitrogen was limiting in the two other scenarios. Furthermore, a substantial amount of microalgal biomass was produced, ranging from 13 to 59 g per person equivalent per day. These findings show that microalgal biofilm systems hold large potential as seasonal wastewater treatment systems and that it is worthwhile to investigate these systems further
Analysis of the contribution of sedimentation to bacterial mass transport in a parallel plate flow chamber Part II: Use of fluorescence imaging
Li, J.Y. ; Busscher, H.J. ; Mei, H.C. van der; Norde, W. ; Krom, B.P. ; Sjollema, J. - \ 2011
Colloids and Surfaces. B: Biointerfaces 87 (2011)2. - ISSN 0927-7765 - p. 427 - 432.
deposition - adhesion - adsorption - biofilms - kinetics - gene
Using a new phase-contrast microscopy-based method of analysis, sedimentation has recently been demonstrated to be the major mass transport mechanism of bacteria towards substratum surfaces in a parallel plate flow chamber (J. Li, H.J. Busscher, W. Norde, J. Sjollema, Colloid Surf. B. 84 (2011)76). Here we describe a novel method for enumerating adhesion of fluorescent bacteria in a parallel plate flow chamber that allows direct imaging of the bacterial distribution along the length of the flow chamber, as caused by sedimentation. Imaging of fluorescence was done using macroscopic bio-optical imaging of the entire flow chamber, including top and bottom plates as well as of the flowing suspension in between. An algorithm is forwarded that allows to separate the fluorescence arising from the suspension and bottom plate and at the same time determines the single cell fluorescence from which the bacterial distribution over the entire bottom plate can be visualized. Enumeration of the numbers of bacteria adhering to the center of the glass bottom plate for a fluorescent Staphylococcus aureus strain was found to coincide with enumerations using phase-contrast microscopy. Moreover, due to the use of macroscopic bio-optical imaging, it was found that the number of adhering staphylococci increases linearly with distance from the inlet of the flow chamber, which could be explained from a simplified mass balance of convection, sedimentation and blocking near the bottom plate of the flow chamber.
Nitrogen and phosphorus removal from municipal wastewater effluent using microalgal biofilms
Boelee, N.C. ; Temmink, H. ; Janssen, M.G.J. ; Buisman, C.J.N. ; Wijffels, R.H. - \ 2011
Water Research 45 (2011)18. - ISSN 0043-1354 - p. 5925 - 5933.
afvalwaterbehandeling - biofilms - anaërobe afbraak - modellen - waste water treatment - anaerobic digestion - models - nutrient removal - photosystem-ii - phytoplankton - growth - bioavailability - photosynthesis - plants - algae - ph
Microalgal biofilms have so far received little attention as post-treatment for municipal wastewater treatment plants, with the result that the removal capacity of microalgal biofilms in post-treatment systems is unknown. This study investigates the capacity of microalgal biofilms as a post-treatment step for the effluent of municipal wastewater treatment plants. Microalgal biofilms were grown in flow cells with different nutrient loads under continuous lighting of 230 µmol/m(2)/s (PAR photons, 400-700 nm). It was found that the maximum uptake capacity of the microalgal biofilm was reached at loading rates of 1.0 g/m(2)/day nitrogen and 0.13 g/m(2)/day phosphorus. These maximum uptake capacities were the highest loads at which the target effluent values of 2.2 mg/L nitrogen and 0.15 mg/L phosphorus were still achieved. Microalgal biomass analysis revealed an increasing nitrogen and phosphorus content with increasing loading rates until the maximum uptake capacities. The internal nitrogen to phosphorus ratio decreased from 23:1 to 11:1 when increasing the loading rate. This combination of findings demonstrates that microalgal biofilms can be used for removing both nitrogen and phosphorus from municipal wastewater effluent
Brushes and proteins
Bosker, W.T.E. - \ 2011
University. Promotor(en): Martien Cohen Stuart, co-promotor(en): Willem Norde. - [S.l.] : S.n. - ISBN 9789085859178 - 142
biofilms - eiwitten - adsorptie - aangroeiwerende middelen - fabricage - biomaterialen - proteins - adsorption - antifouling agents - manufacture - biomaterials

Brushes and Proteins

Wouter T. E. Bosker

Protein adsorption at solid surfaces can be prevented by applying a polymer brush at the surface. A polymer brush consists of polymer chains end-grafted to the surface at such a grafting density that the polymer chains stretch out into the solution. This is schematically shown in figure 1.

Fig. 1. Cartoons of a polymer brush. Two ways of preparation: (a) chemical grafting and (b) grafting through adsorption of block copolymers, for instance by Langmuir-Blodgett deposition (LB).

The main parameters determining the protein resistance of a brush are the grafting density (σ), the chain length (N) and the solvent quality. The thickness of the brush is a function of these parameters: H ~ N σ1/3.

This research is related to biofouling: 'the undesirable accumulation of proteins and cells at a surface', which starts by adsorption of proteins at the surface. Prevention of biofouling is of vital interest in medicine, where bacterial adhesion may cause severe infections on biomaterials used for implants. Treatment with antibiotics has hardly any effect. The only promising remedy against infections in this case is the prevention of a bacterial film. Because protein adsorption is the first step in this process, the research in this thesis is focused on prevention of protein adsorption by polymer brushes.

Numerous studies over the past decades revealed that neutral polymer brushes, especially from poly(ethylene oxide) (PEO), can minimize protein adsorption. Mindful of the parameters determining the adsorbed amount mentioned above, the following three mechanisms can be identified, displayed in figure 2. Primary adsorption occurs when the diameter of the protein is (much) smaller than the distance between the polymer chains. In case of secondary adsorption, the protein is (much) bigger than the distance between the polymer chains. Ternary adsorption results from an attraction between the proteins and the polymer chains in the brush and was first discovered by Currie et al. In 1999. For a considerable time researchers have assumed a repulsion between the proteins and the polymer chains, thereby neglecting the possible ternary adsorption. However, there is increasing evidence that this attraction occurs, especially with PEO brushes. This is highlighted in this research, by adsorption studies at bimodal PEO brushes, consisting of a dense PEO brush of short chains with a varying PEO brush of long chains.

Figure 2.
Different mechanisms for protein adsorption at polymer brushes:

primary, secondary and ternary adsorption.

The main objective of this research was to investigate whether polysaccharide brushes, in particular dextran brushes, could be prepared at a solid surface and to study their protein repellency. It was suggested that brushes from these natural polymers would be more successful to prepare nonfouling surfaces with. Dextran brushes were prepared using Langmuir-Blodgett deposition (LB) and PS-dextran diblock copolymers, illustrated in figure 1. With the LB method it is possible to control both σ and N. The synthesis of the PS-dextran diblock copolymers is described in the thesis as well as the interfacial behavior. Quasi-2D aggregation occurred at the air-water interface during preparation (compression of the PS-dextran monolayer, see figure 1), resulting in inhomogeneous dextran layers at low grafting density. At higher grafting density these aggregates were pushed together to form a homogeneous dextran brush, as illustrated by AFM images. This transition from inhomogeneous to homogeneous results in non-continuous adsorption behavior at dextran brushes, in contrast to PEO brushes, as demonstrated in figure 3.

Figure 3. Normalized adsorption of BSA (Γ/ Γ0) at dextran brushes (■) and PEO brushes (○).

In case of dextran brushes the adsorption of BSA is constant up to a specific σ, followed by a drastic decrease, while PEO brushes show a gradual reduction.Figure 3 also demonstrates that dextran brushes are as efficient as PEO brushes in preventing protein adsorption, at high σ. This is the main conclusion of this research. It is expected that at even higher σ dextran brushes will completely suppress protein adsorption.

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